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ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES BYMATRIX-ASSISTED LASER DESORPTION/IONIZATION MASSSPECTROMETRY: AN UPDATE FOR 2007–2008

David J. Harvey*Oxford Glycobiology Institute, Department of Biochemistry,University of Oxford, Oxford OX1 3QU, UK

Received 22 July 2010; received (revised) 4 January 2011; accepted 4 January 2011

Published online 17 August 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.20333

This review is the fifth update of the original review, publishedin 1999, on the application of MALDI mass spectrometry to theanalysis of carbohydrates and glycoconjugates and bringscoverage of the literature to the end of 2008. The first sectionof the review covers fundamental studies, fragmentation ofcarbohydrate ions, use of derivatives and new softwaredevelopments for analysis of carbohydrate spectra. Amongnewer areas of method development are glycan arrays, MALDIimaging and the use of ion mobility spectrometry.The second section of the review discusses applications ofMALDI MS to the analysis of different types of carbohydrate.Specific compound classes that are covered include carbohy-drate polymers from plants, N- and O-linked glycans fromglycoproteins, biopharmaceuticals, glycated proteins, glyco-lipids, glycosides and various other natural products. There is ashort section on the use of MALDI mass spectrometry for thestudy of enzymes involved in glycan processing and a section onthe use of MALDI MS to monitor products of the chemicalsynthesis of carbohydrates with emphasis on carbohydrate-protein complexes and glycodendrimers. Corresponding anal-yses by electrospray ionization now appear to outnumber thoseperformed by MALDI and the amount of literature makes acomprehensive review on this technique impractical. However,most of the work relating to sample preparation and glycansynthesis is equally relevant to electrospray and, consequently,those proposing analyses by electrospray should also findmaterial in this review of interest. # 2011 Wiley Periodicals,Inc., Mass Spec Rev 31:183–311, 2012Keywords: Carbohydrates; MALDI; Fragmentation; Glyco-proteins; Glycolipids

I. INTRODUCTION

This review is a continuation of the five earlier ones in this serieson the application of MALDI mass spectrometry to the analysisof carbohydrates and glycoconjugates (Harvey, 1999, 2006,2008, 2009, 2011) and is intended to bring the coverage of theliterature to the end of 2008. It is hoped that this review isreasonably comprehensive. However, the current infuriatingpractice of splitting papers between the main text and‘‘supplementary material’’ (e.g., ‘‘Results are shown in Supple-mentary Fig. S1’’), particularly in chemistry journals, means thatsome papers will invariably have been missed if the technique isnot deemed sufficiently important to appear in the main text.

Material not included is research into nucleotides and materialwhere MALDI mass spectrometry has simply been use to recordmasses of glycoproteins. Because the review is designed tocomplement the earlier work, structural formulae etc. that werepresented earlier are not repeated. However, a citation to thestructure in the earlierwork is indicated by its number and a prefixthat refers to the review containing the structure (i.e., 1/x refers tostructure x in the first review and 2/x to structure x in the secondreview).

MALDI continues to be amajor technique for the analysis ofcarbohydrates; Figure 1 shows the year-by-year increase inpapers reporting use of the technique for the period 1991–2008.Reports of carbohydrate analysis involving electrospray ioniza-tion have also increased over this period to such an extent thancompilation of a comprehensive review of this type withoutmulti-author involvement is impractical. However, most of thework described in this review relating to sample preparation andglycan synthesis is equally relevant to electrospray and,consequently, those proposing analyses by electrospray shouldalso find material in this review of interest. Possibly one of themain advantages ofMALDI for glycan analysis is that it producesexcellent glycan profiles of mixtures of neutral glycans becauseof the tendency of the technique to produce only [MþNa]þ ions(possibly accompanied by small amounts of [MþK]þ) unlikeelectrospray that tends to give spectra containing ions in differentcharge states as well as ions arising from different adducts. Onthe other hand, MALDI suffers from problems with sialylatedglycans that are generally unstable under MALDI conditionsnecessitating use of specialized conditions or derivatization forobtaining satisfactory spectra. Nevertheless, the speed andconvenience of MALDI analysis still makes it the method ofchoice in many situations.

General books, reviews, and review-type articles directlyconcerned with, or including MALDI analysis of carbohydratesor glycoconjugates to have been published during the reviewperiod include a book on MALDI MS (Hillenkamp & Peter-Katalinic, 2007), general reviews by Harvey (2007), Sagi andPeter-Katalinic (2007), Zaia (2007), and by Pilobello and Mahal(2007) (brief, 40 references). Other relevant reviews includethose on carbohydrate analysis by mass spectrometry (Sekiya &Iida, 2008), mass spectrometry of carbohydrates (Kamerlinget al., 2007; Rodrigues et al., 2007b), carbohydrate chemistry(Lindhorst, 2007), recent developments on MALDI-TOF ofbiological samples (Pan et al., 2007), sample preparation(particularly for chromatography, Luque-Garcia & Neubert,2007; Sanz & Martınez-Castro, 2007), quantitative aspects ofMALDI-TOFMS (63 references) (Duncan, Roder,&Hunsucker,2008) and MALDI-ion-trap mass spectrometry (Matamoros

Mass Spectrometry Reviews, 2012, 31, 183– 311# 2011 by Wiley Periodicals, Inc.

————*Correspondence to: David J. Harvey, Oxford Glycobiology Institute,

Department of Biochemistry, University of Oxford, Oxford OX1 3QU,

UK. E-mail: [email protected]

Fernandez, 2007). A major book on glycobiology has beenpublished (Varki et al., 2008) but it contained a disappointinglysmall amount of mass spectrometry. Other books of interestinclude Watson and Sparkman’s (2008) ‘‘Introduction to MassSpectrometry’’ and the two volume ‘‘Experimental Glycoscien-ces’’ containing many experimental details for sample prepara-tion, etc. (Taniguchi et al., 2008). Other more specialized reviewsare cited below in the sections to which they belong.

II. THEORY

Although theMALDI techniquewas invented about 20 years ago,there is still debate on how the ions are formed. A study byChanget al. (2007b) proposes a pseudo-proton transfer process duringcrystallization as a primary mechanism for producing analyteions. The authors propose an energy transfer-induced dispro-portionationmodel to explain the observation of an equal amountof positive and negative ions produced in MALDI for largebiomolecules.

Electron affinities for seven commonMALDImatrices havebeen published (Lippa et al., 2007) but, although the informationwill be valuable for development of models of the MALDIprocess, the authors point out that equivalent data for the analytemolecules is also necessary. Potassium cation affinities ofbenzoic acid derivatives have been determined by thresholdcollision-induced dissociation (Chinthaka & Rodgers, 2007).With 2-hydroxy substituted benzoic acids, as in 2,5-dihydroxy-benzoic acid (DHB, 1/26), the potassium ion forms a 4-membered ring with the two oxygen atoms of the carboxylicmoiety that is stabilized by hydrogen bonding from thehydroxylic hydrogen atom. Heaton and Armentrout (2008) havemeasured the binding affinities between sodium and severalmonosaccharides; the overall trend was found to be b-Ara< a-Ara< b-Xyl< b-Glc<a-Glc< a-Xyl<a-Gal< b-Gal.

III. MATRICES

A review describing the search for the perfectMALDImatrix hasbeen published (Batoy et al., 2008) and Najam-ul-Haq et al.

(2007) have reviewed the use of carbon nano-materials asmatrices. Erra-Balsells and Nonami (2008) have compared therelative performance of nor-harmane (1/35) with classicalMALDI matrices.

A. Theory of Matrix Action

Another comparison of the ability of dihydroxybenzoic acidisomers to act as matrices, this time with lipids, has again foundthe 2,5-isomer to be the best. Decreasing efficiency of the otherisomers fellwith their acidity (Schiller et al., 2007). Jessome et al.(2008) have compared signals from several isomers of DHB andconcluded that neither thermodynamic properties nor gas-phaseprotonation mechanisms could explain the observed results.However, the formation of [MþNa]þ ions from carbohydrateswas not mentioned.

B. Simple Matrices

Oxidized carbon nanotubes (CNTs) with short and open-endstructures have been shown to produce strong signals from smallcarbohydrates and amino acids (Wang et al., 2007a). The CNTs,prepared by ethylene vapor deposition onto a porous anodicalumina (PAA) template, were washed and suspended in water/methanol and deposited onto the MALDI target plate. Themethanol evaporated, the sample solution was added and themixture allowed to dry under ambient conditions. MALDIspectra were recorded with an Fourier transform (FT) instrumentfitted with a nitrogen laser. Ions were generally [MþNa]þ fromthe carbohydrates such as mono- and di-saccharides. By usingthis method, the background interference signals generallycaused by amorphous carbon powder in CNTs could be reducedand it was shown that the signal intensity of the CNTs preparedwith a PAA template was much lower than that of commercialmulti-wall carbon nanotubes.

Carbon nanotubes have also been shown to act as veryefficient MALDI matrices for small carbohydrates in planttissues. Thus, Gholipour, Nonami, and Erra-Balsells (2008a)have investigated two sample preparation methods with tissuefrom tulip bulbs and leaves. First, the carbon nanotubes weredeposited on fresh tissue slices placed on the probe and, inthe second method, semitransparent tissues were placed on adried layer of nanotubes on the probe. Spectra recorded inpositive ion mode gave the best results. With the tissue placedover the nanotubes, virtually no matrix ions were seen, thus thismethod proved to be excellent for ionization of mono-saccharides and other small sugars. Amyloglucosidase degrada-tion of starch was achieved on the tissue surface and the liberatedglucose was observed with the aid of the nanotubes. In anotherpublication (Gholipour, Nonami, & Erra-Balsells, 2008b), theseauthors used a pressure probe to extract cytoplasm from singlecells of the same two tissues and examined it by MALDI-TOFMSwith DHB, 2,4,6-trihydroxyacetophenone (THAP, 1/44) andcarbon nanotubes. DHB produced mainly [MþNa]þ ionswhereas [MþK]þ ions were dominant from the other twomatrices. Oligosaccharides were detected to 15 hexose residueswith THAP, 11 with DHB but only 7 with the nanotubes.

The presence of abundant ions derived from the matrixfrequently limits the usefulness ofMALDIMS for the analysis ofsmall molecules. To overcome this problem, Zhang et al. (2008b)have used acidic fullerene (C60CHCOOH) as the matrix and

FIGURE 1. Papers reporting the use of MALDI MS for the analysis of

carbohydrates and glycoconjugates by year.

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184 Mass Spectrometry Reviews DOI 10.1002/mas

obtained spectra from disaccharide propyl glycosides. Anotherrelated method for obtaining spectra of small molecules is to usecolloidal graphite rather than an organic matrix. The technique,developed by Zhang, Cha, and Yeung (2007a) and namedgraphite-assisted laser desorption/ionization (GALDI) consistsof simply coating the MALDI target plate with the colloidalgraphite solution, allowing this to dry and then applying thesample. GALDI spectra of fatty acids, flavonoids and carbohy-drates were obtained in negative ion mode ([M�H]� ions).Carbohydrates from mono-to tetrasaccharides gave strongspectra but the molecular ions were accompanied by smallamounts of [M–H–H2O]

� and ring-cleavage fragments. Themethod was used to study the composition of fruit juices and formeasuring the distribution of small molecules in tissues byGALDI-imaging (see below).

A study of pencil ‘‘lead’’ (amixture of graphite and clay) as amatrix for small molecules has concluded that the soft 2B pencilsgive the best spectra (Langley, Herniman, & Townell, 2007).Little or no matrix contamination was present in the spectra andstrong cationic species, predominantly [MþK]þwere observed.Although sugars were successfully analyzed, no details werereported for ‘‘confidentiality reasons.’’

A study by scanning microprobe matrix-assisted laserdesorption/ionization mass spectrometry (SMALDI MS) ofdried droplet preparations of DHB have produced massspectrometric images of lateral distributions of sample compo-nents and impurities within and upon the crystals with a lateralresolution of 1 mm. The results showed inhomogeneous separa-tion of components on various scales between millimeters andsub-micrometers. Peptide ion signals were observed from insidethe matrix crystals, while carbohydrate signals and alkali ionsignals were observed predominantly from outside the largermatrix crystals. This distribution accounts for the frequentlyobserved rapid fading of the MALDI signal from carbohydratesrecorded from DHB (Bouschen & Spengler, 2007). A study ofsample preparation techniques with DHB has shown a consid-erable improvement in signal intensity, as much as 10-fold forNeu5Ac (1/11), if the sample is actively dried. Crystals producedin this manner are smaller than for passively dried samples.Signal intensity was also increased if 10mM sodium chloridewas added to the matrix (Williams et al., 2007).

Fenaille et al. (2007a) have shown that THAP is a moreefficient matrix than the more commonly used sinapinic acid forionization of glycoproteins, even those with masses as high as150 kDa. Singly charged ions were the major species producedbut the larger glycoproteins yielded both doubly and triplycharged ions in addition. An extensive study of this matrix hasbeen reported by Hsu et al. (2007d) who found it to be valuablefor ionization of large polysaccharides and protein-carbohydratecomplexes. For example, resolved mixtures of PL-23k poly-saccharides in the mass range 12–20 kDa were obtained and astrong peakwas obtained frombovine serumalbumin (BSA)with51 synthetically attached mannose residues (78,674Da).

In a comparative study of several common matrices (nor-harmane, DHB, THAP, 2-(40hydroxyphenyl)azobenzoic acid(HABA, 1/32), indoleacrylic acid (IAA, 3/5), 6-azo-2-thiothy-mine (ATT, 1/45), and 5-chloro-2-mercaptobenzothiazole(CMBT, 1/33)) with acetylated monosaccharides, the best resultswere obtained with nor-harmane and DHB using the sandwichpreparation method but with DHB producing the strongestsignals. THAP gave weaker but similar quality signals with no

cluster or satellite ions whereas IAA gave strong cluster andsatellite signals. ATTand CMBTare mild acids and were used incombination with the stronger acid, DHB, to see if desorption/ionization efficiency could be better than with DHB alone. Nosignificant improvement was observed. HABA produced spectraonly from acetylated glycosylamines (Sato et al., 2007). Xie et al.(2007) have shown equivalent performance with N-glycans withDHB and arabinosazone (1/40). In another comparative study ofDHB, THAP, ferulic acid and a-cyano-4-hydroxycinnamic acid(CHCA, 1/23) for the analysis of fructooligosaccharides used asfeed additives, DHB again proved to be the most satisfactory(Reiffova et al., 2007). As the tables in the following sectionsshow, DHB is by far the most common matrix for most types ofcarbohydrate.

Coumarin (1) and the substituted coumarins, 3-hydroxy-coumarin (2), 3-aminocoumarin (3), 3-carboxycoumarin (4) and4-methyl-7-hydroxycoumarin (5) have been tested for theirability to ionize carbohydrates and glycoproteins (Zhang, Deng,& Deng, 2008h). The best results for dextran were obtained froma mixture of coumarin and DHB or of 3-hydroxycoumarin andDHB. The coumarins themselves, particularly 3-hydroxycou-marin, 4-methyl-7-hydroxycoumarin and 3-aminocoumarinwere effective in ionizing the glycoproteins.

ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES &

Mass Spectrometry Reviews DOI 10.1002/mas 185

C. Binary Matrices

A novel DHB/N,N-dimethylaniline (DHB/DMA) matrix hasbeen developed for detection and quantitative analysis ofnative N-linked oligosaccharides (Snovida & Perreault, 2007).Substantial improvements in sensitivity were observed relativeto the signals obtained with a traditional DHB matrix. Thematrix crystal layer was more uniform than that normallyobtained fromDHB allowing reproducible and consistent massspectra to be obtained without spot-to-spot variations in signalstrength. The matrix was shown to be suitable for quantitativework with detection limits in the fMole range. Snovida, Rak-Banville, and Perreault (2008) have also investigated theproperties of DHB combined with aniline and compared theresults with those from the DHB/DMA mixture. Both matricesgave superior spectra to DHB alone and produced a morehomogeneous target. Aniline formed a Schiff base withreducing sugars on the MALDI target that could be useddiagnostically to identify reducing sugars. DMA did notundergo this reaction.

The binary matrix, 8-hydroxyquinoline (8-HQ, 6) andDHB has been reported to be a good matrix for glycoproteins(Zhou & Deng, 2008). 8-HQ itself was a poor matrix but,when mixed with DHB, it caused much finer crystals to formand to produce better sample-to-sample reproducibility andsignal-to-noise ratios than when DHB was used alone.Soltwisch, Berkenkamp, and Dreisewerd (2008) have mixedDHB with glycerol in the ratio of 1:5 and produced excellentspectra from small oligosaccharides and peptides. Sample spotswere relatively homogeneous and were prepared from thesample/matrix mixture on-target by first evaporating most ofthe solvent with a stream of air and then, in order to evaporatethe rest of the solvent, the sample plate was placed in arough vacuum At approx. 10�2 mbar. Full crystallization wastypically observed after 45–90min with crystals that rangedin length from about 100 to 300 mm and with a width of about15–30 mm.

The energy-absorbing substances aminopyrazine (AP, 7),4,40-azodianiline (ADA, 8), and 1-chloro-4-hydroxyisoquinoline(CHIQ, 9) have been evaluated as MALDI matrices andshown to be valuable for examination of small carbohydratesbecause of the virtual absence of matrix ions in the lowmass region. Excellent results were also achieved with thesecompounds in combination with existing acidic matrices.A combination of DHB and AP in the ratio of 3:1 wasparticularly effective. Analytes could be detected to 4 fmol/mL.The matrix was also effective for cyclodextrins, maltooligo-saccharides and dextrans with the latter giving ions fromcompounds as high as Glc40 (Hashir, Stecher, & Bonn,2008).

A combination of THAP and a-cyclodextrin (4/24) in a 1:1molar ratio has been used to ionize compounds such as substanceP and chitobiose (GlcN-b-(1! 4)-GlcN). The presence of thecyclodextrin caused total suppression of the matrix ions and alsoproduced [MþH]þ ions from the carbohydrates (Yamaguchiet al., 2008b). Grant and Helleur (2008) have found that thesurfactant cetyltrimethylammonium bromide (10), whenadded to CHCA or THAP, considerably reduced the abundanceof the matrix ions. The analyte ions were also reduced but toa much lesser extent. Using the THAP-surfactant, the inves-tigators were able to quantify anthocyanins in various berrysamples with standard deviations of less than 10%, greatlyreducing the analysis time from that of the traditional LC/MSmethods.

D. Liquid Matrices

The guanidinium (11) salt of CHCA has been shown to be apromising liquid matrix for analysis of sulfated carbohydratessuch as dermatan sulfate and chondroitin sulfate because of itsvery low tendency for causing sulfate loss (Laremore, Zhang, &Linhardt, 2007). The 1,1,3,3-tetramethylguanidinium (TMG, 12)salt of CHCA (G2CHCA) was reported by Laremore, Zhang, and

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Linhardt (2007) in 2007 as a useful ionic liquid matrix forsulfated oligosaccharides because it suppressed the loss of sulfategroups. Fukuyama et al. (2008) have now compared this matrixwith DHB and a new ionic matrix, the TMG salt of p-coumaricacid (13, G3CA) for both positive and negative ionization ofsulfated and phosphorylated glycans. Sulfated oligosaccharideswere detected with high sensitivity (e.g., 1 fmol) in both ionmodes and the dissociation of sulfate groups was suppressedparticularly with G3CA. Sialylated and neutral oligosaccharideswere also detectedwith high sensitivity (1 fmol) with positive ionextraction and little loss of sialic acid. The use of 1-ethyl-imidazolium (14) a-cyano-4-hydroxycinnamate (ImCHCA)for the analysis of glycosaminoglycans (GAGS) is describedbelow.

E. Other Matrices

Aquatic fulvic acid, a complex mixture of organic compoundsbelonging to the poorly characterized class of compounds knownas humic substances has been shown to be an excellent matrix forsmall organic molecules including carbohydrates with adetection limit of about 10 mg/mL (Mugo & Bottaro, 2007).

As with conventional matrices, carbohydrates typicallyyielded [MþNa]þ ions. Few, if any matrix-related ions wereseen except when the analytes were present in very lowconcentration.

Zinc sulfide nanoparticles capped with various functionalgroups have been investigated as combined affinity probes andmatrix, with cyclodextrins (CDs) and proteins used as the testcompounds (Kailasa, Kiran, &Wu, 2008). The best results wereobtained with 3-mercaptopropanoic acid (15) as the cappingagent. To record the spectrum, the nanoparticles weremixedwiththe carbohydrate solution, the pH was adjusted (pH 7 wasoptimum for the CDs) and the solution was vortexed at 900 rpmfor 30min. Samples were transferred to the MALDI target andallowed to air-dry. Detection limits of 20–55 nM were reportedfor the CDs.

F. Matrix-Free Systems

Porous alumina coated with platinum has been found to produceabundant ions from a variety of compounds including glucose(in 10mM NaCl) and ribonuclease B (Wada, Yanagishita, &Masuda, 2007a). The platimum layer (5–20 nm) apparentlymelted under the influence of the laser beam (337 nm) but thetarget remained active for several months in air.

A one-way multilayered flexible hydrophobic solutionrepellent foil named DropStopTM (hard lacquer, metal layer,PET, metal layer, hard lacquer), obtained from a commercialsupplier or the local wine store has been fixed to a standardMALDI target for normal UV MALDI-TOF experiments or forautomated off-line coupling of capillary zone electrophoresis(CZE) with MALDI. Various matrices and sample preparativetechniques were evaluated with regard to matrix suppressioneffects, sensitivity, crystallization of deposited samples and theextent of drop migration on the surface. The results werecompared with those obtained on conventional stainless steeltargets. The foil was found to give reduced chemical noise andspot area and could be used for small molecules, carbohydrates,and proteins (Rechthaler, Rizzi, & Allmaier, 2007).

In order to avoid problems with matrix ions duringthe analysis of small molecules including carbohydrates,Hashir et al. (2007) have introduced a matrix-free system termedmatrix-free material-enhanced laser desorption/ionization MS(mf-MELDI). The system consists of 4,40-azo-dianiline (8)immobilized on silica gel with optimized particle and pore sizesfor absorption of the laser energy. To record a spectrum, a thinlayer of the material, suspended in methanol, was applied to aconventional stainless steel target and allowed to dry. The analytesolution was then placed on the surface and spectra wererecorded, as normal, with a UV laser. The system producedstrong, matrix-free spectra from monosaccharides such asglucose and xylose with a detection limit for xylose of 70 fmoland was applied to the analysis of wheat straw degradationproducts and to carbohydrates from the bark of the white willow(Salix alba L.).



Gold nanoparticles have shown promise for analysis ofsmall carbohydrates and have the advantage of the absenceof the prominent matrix peaks that are common with matricessuch as DHB. The technique, termed surface-assisted laserdesorption/ionization mass spectrometry, or SALDI-MS, pro-duced [MþNa]þ ions and was used to measure glucose in urinein the low nM range (Su & Tseng, 2007). Wu et al. (2007b) haveused what they term the ‘‘sample first method’’ to prepare thetarget. The sample was deposited first, allowed to dry and thenthe solution of gold nanoparticles was added. The results fromthis preparative method were better than those obtained from thereverse procedure or from the dried droplet method. Initialstudies were performed with cyclodextrins but the method alsogave good results with other small carbohydrates, steroids, andpeptides.

The MALDI matrices CHCA and DHB have beenconjugated to magnetic nanoparticle to give MNP@matrix thatprovides high ionization efficiency free of background peaks andwhich is excellent for small molecules such as monosaccharides(Lin et al., 2007). Further conjugation of proteins providesa system capable of affinity extraction and ionization. Thus,mannose was successfully extracted and analyzed from a spikedserum sample with MNP@DHB-ConA by magnetic removal ofthe bound monosaccharide to give a MALDI-TOF spectrumcontaining essentially only ionized mannose after simply wash-ing the nanoparticles three times with sodium bicarbonate buffer.Without extraction the mannose peak was hardly discernable.

G. Matrices Producing Negative Ions FromNeutral Glycans

Neutral glycans do not usually form negative ions from thecommon matrices such as DHB but can be induced to do so withmatrices such as nor-harmane that have gas-phase basicitieslower than or close to HCl. Alternatively, some investigatorshave adducted carbohydrates with anions such as chloride andsulfate and observed strong signals. Chmelık et al. (2007) haveinvestigated the addition of ammonium chloride, nitrate, hydro-gen–carbonate and hydrogen–sulfate to DHB or THAP andreport that hydrogen–carbonate appears to produce the mostintense [MþH]� ions without adduct formation. The mostabundant adducts were formed with nitrate ([MþNO3]

�). Theinfluence of the matrix was not reported. Tri- to hexa-mers fromthe terminal epitope of the O-antigen from Vibrio cholerae O:1serotypes Ogawa and Inaba were used as the target glycans.Fragmentation with a TOF/TOF instrument produced a cleavagereaction of the penultimate sugar ring to give an ion withthe structure Sugar–O–CH¼CH–O� of the type observedearlier from N-glycans (Harvey, 2005a; Harvey et al., 2008a).However, in this case, the electron shift resulted in retention of thecarbon atoms at positions 2 and 3 as the result of linkage at C2rather than at C4 as in the N-glycans. The negative ion MALDI-TOF/TOF fragmentation spectra were reported to be simplerthan the positive ion spectra but, at the same time, to be moreinformative.

A mixture of harmine (1/34) and ammonium chloride hasproved to be a useful matrix for producing negative [MþCl]�

ions from neutral carbohydrates (Yamagaki, Suzuki, & Tachi-bana, 2007). The relative abundances of the [MþCl]� ionscorrelated with the amount of NH4Cl added and saturated whenthe NH4Cl concentration was approximately four times that of

the matrix. Harmine hydrochloride was thought to be the actualmatrix. Negative ion MALDI spectra of sulfated glycosphingo-lipids have also been obtained from nor-harmane but, in this case,[M�H]� ions were formed by ionization of the sulfate group(Landoni et al., 2007)

IV. IR MALDI

The utility of atmospheric pressure IRMALDImass spectrometryusing the instrument described earlier (Li, Shrestha, & Vertes,2007g) has been assessed for plant metabolomic studies (Li,Shrestha, & Vertes, 2008f). Tissue sections from plant organs,including flowers, ovaries, aggregate fruits, fruits, leaves, tubers,bulbs, and seeds were studied in both positive and negative ionmodes. Water in the plant material acted as the matrix. For leaves,single laser pulses sampled the cuticle and upper epidermal cells,whereas multiple pulses were demonstrated to ablate somemesophyll layers. Positive ion mode yielded mainly [MþK]þ,[MþH]þ or sometimes [MþNa]þ ions, whereas [M�H]� ionsdominated the negative ion spectra. Over 50 smallmetabolites andvarious lipids nucleosides and carbohydrates were detected. Theuse of a 3mm mid-IR laser with thiourea as the matrix has beenshown to significantly reduce the amount of in-source fragmenta-tion of glycopeptides and 2-aminopyridine (2-AP, 1/52)-labeledcarbohydrates (Takahashi, 2008).

V. DESORPTION ELECTROSPRAYIONIZATION (DESI)

A comparative study of desorption electrospray ionization(DESI) and MALDI using O-linked glycans has shown that thetwo techniques produce almost identical spectra even thoughthe ionization conditions are very different (liquid vs. solidand atmospheric pressure vs. vacuum) (Bereman, Williams, &Muddiman, 2007).

VI. MALDI IMAGING

Imaging using MALDI mass spectrometry is a growing area andone that offers great potential for location of a variety of analytes.In the carbohydrate field, glycosphingolipids have been a majortarget. One of the problems associated with the technique islateral diffusion of the analytes following matrix application andthe consequent loss of resolution. Several methods have beendeveloped to overcome the problem. One method is to dispensewith thematrix and ionize the samplemolecules directly from thetissue. Thus, Dreisewerd et al. (2007) have used an IR laserdesorption ionization orthogonal time-of-flight mass spectrom-eter (IR-LDI-o-TOF-MS) to obtain molecular ion profilesdirectly from native tissue and from whole oils from plants andanimals. The instrument used an Er:YAG laser (2.94 mm)emitting pulses of about 100 nsec in duration at a maximumrepetition rate of 2Hz. The laser beam irradiated the sample at anangle of incidence of about 558 relative to the sample platenormal and produced a spot size of about 100mmin diameter. Theions were produced in an elevated pressure ion source filled withnitrogen gas (0.1�1mbar in the sample plate region) and wereaccelerated into a quadrupole ion guide filled with nitrogen at apressure of about 10�3mbar. Dry samples were attached to thesample plate and examined directly. The absence of a matrixavoided problems with analyte diffusion but limited the mass

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range to less than 2 kDa, much lower than could be obtained withthe use of a matrix. Compounds such as carbohydrates,phospholipids, triglycerides, and flavonoids were readilyobserved but peptides and proteins gave weak signals. Themethod was used to examine a variety of samples includingseeds, coconut, pork meat, strawberries, canned green olives,rose leaf, and various oils.

Another method, developed by Chen et al. (2008f) used anew oscillating capillary nebulizer matrix coating system thatsprayed small droplets of matrix (DHB) aerosol onto the samplesurface. The distance between the nozzle and the sample wasabout 12 cm and multiple matrix coating cycles with discretespraying and drying were performed to get the best imagingresults. Typically, the spraying and drying time for a 10� 10mm2

sample was 10 and 30 sec, respectively and usually about 30coating cycles were required to provide an optimal matrixthickness of 5 to about 50 mm. The system has been used to imageglycosphingolipids in histological slices of brains from a mousemodel of Tay-Sachs and Sandhoff disease. MALDI imaging hasbeen used to determine the sites of neutral glycosphingolipidaccumulation in skin and kidney tissue of Fabry disease(a-galactosidases A deficiency) patients (Touboul et al., 2007).Co-localization was found with cholesterol and vitamin E.

Wang et al. (2008a) have compared a thin-layer chromatog-raphy (TLC) sprayer, a Collision Nebulizer, and an artisticairbrush for their suitability in applying the matrix. Althoughwidely accepted as a matrix deposition method for MALDI-IMSapplication, the TLC sprayer appeared to generate coarse andunevenly sized droplets in spite of careful adjustment of theairflow and pressure of the nebulizing gas. The CollisionNebulizer, on the other hand, generated a fine mist that was verysuitable formatrix depositions. Unfortunately, most of thematrixwas deposited onto the tissue-free region of the target plate and,furthermore, the spraying process consumed excessive amount ofmatrix solution. Such inefficiency prolonged the spraying timefor over 1 hr in order to cover a brain section with enough matrixand exposed the tissue section to room temperature for anexcessive time. Use of the airbrush seemed to be a compromise ofthe above two methods and offered several advantages. Itappeared to generate a more homogenous droplet size than theTLC sprayer and rarely caused uneven matrix deposition.Further, matrix deposition by airbrush could be carried out ina timely manner without consuming excessive amount of matrixsolution. When the distance between the target plate and theairbrush nozzle was properly adjusted, airbrush spraying did notcause surface over-wetting as was cautioned by other inves-tigators. It was used to map several phospholipids and sulfatides(16) in rat brains.

Airbrush application of a DHB matrix was also usedby Shimma et al. (2008) to demonstrate that the ShimadzuAXIMA-QIT instrument can effectively be used for imagingexperiments and to provide MSn spectra. The method was usedto map phospholipids, glycolipids and peptides in mousecerebellum sections. An airbrush was also used by Cha et al.(2008) to spray colloidal graphite dissolved in 2-propanol ontoplant surfaces and stem cross-sections of Arabidopsis thaliana ina technique termed graphite-assisted laser desorption/ionizationor GALDI (see above). This technique enabled images ofcarbohydrate and carbohydrate-containing compounds suchas flavonoids to be obtained with good resolution. Tissue-specific accumulations of flavonoids in flowers and petals wereobserved.

The relative distribution of gangliosides with C18 orC20 sphingosine chains has been examined in male mousebrains by Sugiura et al. (2008b). Frozen brain slices werethaw-mounted on indium-tin oxide glass slides or sheets andDHB was sprayed on with an airbrush. Both QIT-TOF andTOF/TOF spectra were acquired. The results showed that,although the C18 species was widely distributed, the C20species selectively localized along the entorhinal-hippocampusprojections, especially in the molecular layer of the dentategyrus.

Shroff et al. (2008) have used MALDI imaging to showthat glucosinolates (17) are more concentrated in the midveinand periphery of A. thaliana leaves than in the inner lamellaand that this explains the feeding pattern of Helicoverpaarmigera (cotton bollworm) larvae that prefer to avoid theseregions. To obtain the spectra, leaves were sprayed with thematrix, 9-aminoacridine (18) using an airbrush with an 0.2mmnozzle. Spraying continued for 20–22 sec, after which time theleaf was allowed to dry for 5min. The process was typicallycarried out 15 times for maximum signal strength. MALDI-TOFspectra were acquired with a bench-top instrument fitted with anitrogen laser.



Vertes et al. (2008) have usedMid-IR (Nd:YAG laser drivenoptical parametric oscillator, 2,490 nm) to image the letters ‘‘IR’’written on paper with a pencil. The monitored ion at m/z 365corresponded to the [MþNa]þ product from a disaccharide.Additional examples of MALDI imaging are detailed below.

VII. DERIVATIVES

A. Reducing Terminal Derivatives

Reducing terminal derivatization is most frequently used foradding fluorophores to carbohydrates andMALDI-TOF analysisoften finds application in confirming the structures.

1. Reducing Terminal Derivatives Prepared byReductive Amination

MALDI-TOF analysis has been used to characterize 3-amino-9-ethylcarbazole (19) derivatives prepared asUVadsorbing tags forHPLC analysis of monosaccharides. The compounds formed[MþH]þ ions from DHB rather than the more usual [MþNa]þ

species (Zhang,Huang,&Wang, 2007e).Amethod for gas-phasepreparation of fluorescent 2-AP derivatives that avoids the needfor removal of the excess of reagent has given glycan profiles(from chicken ovalbumin) comparable to that obtained bytraditional liquid-phase preparation (Nakakita et al., 2007b).The glycanswere deposited in thewall of amicrotube and reactedunder reduced pressure with vapors (1008C) from the reagentand acetic acid. The reducing agent, a dimethylamine-boranecomplex was then added to another tube and the sealed reactionvessel was again heated to 1008C but at a lower vacuum (40 kPa).Excess reducing agent was removed with toluene.

2. Reducing-Terminal Derivatives Prepared byOther Methods

The traditional procedure for preparing 1-phenyl-3-methyl-5-pyrazolone (5/7) derivatives has been modified by using liquidammonia instead of sodium hydroxide as the base (Wang et al.,2007d). The derivatives could be analyzed directly by MALDI-

TOFwithout a desalting procedure.Xyloglucan oligosaccharideshave been fluorescently labeled with sulforhodamine B (20) forassessment of transglycosylating activity of plant xyloglucanendotransglucosylase/hydrolase (Kosık & Farkas, 2008).The starting xyloglucan-derived oligosaccharides were firstconverted to their corresponding 1-amino-1-deoxyalditols (glyc-amines) by incubation with ammonium acetate and NaCNBH3

at 808C for 2–4 hr, and these compounds are then reacted withLissamine rhodamine B sulfonyl chloride to obtain fluorescentlylabeled derivatives. Products were monitored by MALDI-TOFMS.

On-target derivatization with pyrenebutyric acid hydrazide(PBH, 21) with DHB as the matrix has been reported to increasethe signal strength of keratin sulfate oligosaccharides making itsuitable for trace analysis (Zhang et al., 2008g). To form thederivative, the keratin sulfate was dried on the target and asolution of PBH, acetic acid andMeOHwas added. This mixturewas heated at 608C for 10min, cooled to room temperature andthe matrix solution was added. MALDI spectra were acquiredwith a TOF/TOF instrument allowing fragmentation spectra to beobtained.

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Gil, Kim, and Kim (2008) have developed a methodfor identification and quantification of neutral, enzymaticallydesialylated N-glycans that involves derivatization with Girard’sT reagent (1/55) to introduce a fixed cationic charge in orderto overcome peaks formed from multiple cation adduction([MþNa]þ, [MþK]þ, etc.). Results agreed with those obtainedby HPLC but the resolution and sensitivity were higher, allowingmore minor constituents of mixtures to be determined. Themethod was applied to the analysis of N-glycans from IgG,chicken ovalbumin and miniature pig kidney glycoproteins.

B. Derivatives of Other Sites

1. Permethylation

An improved method for permethylation of carbohydratesdirectly from aqueous solution by the sodium hydroxide/NaOHmethod was been reported (Ciucanu & Caprita, 2007). Theaddition of an excess of solid NaOH at a concentration of4mg/mL of water provided sufficient desiccant activity to give a98% yield with no by-products.

Kang, Mechref, and Novotny (2008c) have published anextension to their capillary permethylation technique (Kanget al., 2005) that utilizes spin columns packed with sodiumhydroxide beads. The method was said to offer simplicity andreproducibility allowing complete permethylation of 12–18samples in less than 20min. The method gave identical results tothose from the capillary method as shown by MALDI-TOFprofiles of N-glycans from human serum glycoproteins, a1-acidglycoprotein and ribonuclease B.

Price (2008) has used MALDI-TOFMS to compare the twostandard permethylation techniques using dimsyl base or sodiumhydroxide with methyl iodide for permethylation of b-cyclo-dextrin (4/6). Unlike the dimsyl base, sodium hydroxideproduced incomplete permethylation and a spectrum thatcontained a series of ions spaces 14 mass units apart below thatof the fully permethylated compound. The 3-hydroxy groupswere found to be the ones undergoing incomplete methylation.

2. Pertrimethylsilylation

Reaction of small oligosaccharides with trimethylsilylimidazole(22) and examination of the products byMALDI-TOF fromDHBhas given the molecular ions of the per-trimethylsilylated (TMS)carbohydrates and a series of ions separated by 72 mass unitsproduced either by incomplete reaction or partial hydrolysisenabling the number of hydroxyl groups in the carbohydrate to beenumerated (Adeuya & Price, 2007).

3. Stabilization of Sialic Acids by Derivative Formation

Sialic acids have been stabilized for MALDI analysis byesterification of the labile acidic protons of the carboxylic acid.Thus, Liu et al. (2007b) have used the reaction of methyl iodidewith the sodium salt of carboxylic acids to form methyl esters ofsialylated glycans obtained by pronase E digestion of glyco-proteins. This proteolysis reaction leaves asparagine attached tothe carbohydrate, which, in the presence of the methyl iodide isconverted into its trimethyl quaternary ammonium salt. The acidof the asparagine residue is simultaneously converted into itsmethyl ester. The reaction was reported to increase the detectionlimit by 10-fold and was applied to glycans from ribonuclease B,ovalbumin, and transferrin.

The reaction of sialylated glycans with 3-methyl-1-p-tolyltriazene (MTT, 23) in a mixture of DMSO and acetonitrileat 608C for 1 hr has been shown to give excellent formation of themethyl esters (Miura et al., 2007b). As an extension to themethod, the authors released N-glycans from reduced andalkylated porcine fibrinogen with PNGase F and then immobi-lized them on an Affi-Gel Hz (BioRad) hydrazide-type resin.After being washed, the glycans were methylated with MTT(608C, 2 hr), the esterified glycans were released with trifluoro-acetic acid (TFA) and converted to 2-aminobenzamide (2-AB, 1/56) derivatives for analysis by HPLC and MALDI-TOF MS.Good quantitative correlation between the two techniques wasobserved. The methylation method was also shown to work withglycans attached to gold colloidal nanoparticles.

An alternative approach for sialic acid stabilization is tocreate internal esters (lactones) and this approach has been usedby Galuska et al. (2007) to analyze oligo- and poly-sialic acids,compounds that are widespread in many organisms. They occurin humans attached to the N-glycans of neural cell-adhesionmolecule. Quantitative acid-catalyzed lactonization wasachieved with TFA and o-phosphoric acid and, of severalmatrices tested,ATTproved to be themost satisfactorywithDHBbeing nearly as good. When sialic acids were a-(2! 8)-linked,cyclisation with the hydroxyl group at C-9 of an adjoining sialicacid residuewas used, whereas with a-(2! 9)-linked poly-sialicacids, cyclisation occurred with the hydroxyl group at C-8.Because, lactonization resulted in precipitation of the product,reactions were performed on the MALDI target. Good signal-to-noise ratios were obtained even with masses greater than 10,000and polymers with up to 100 residues could be examined. Unlikethe a-(2! 8)-linked sialic acids that reacted readily under theabove conditions, a-(2! 9)-linked acids were much morereluctant to form lactones, thus providing a method for differ-entiating the two linkages.

Because of difficulties that have been noted in methyl esteror amide formation from the acid group of a2! 3-linked sialicacids, Toyoda et al. (2008) have investigated alternative methodsof derivatizing these groups and have produced a method using



acetohydrazide (24) derivatization. The reaction appeared to bequantitative with both a2! 3- and a2! 6-linked sialic acidsas demonstrated with N-glycans released from bovine fetuin, aglycoprotein containing sialic acids in both linkages. Themethodwas, however, not appropriate for glycans with an intact reducingterminus because the reagent also reacts with aldehydes. Thus,the glycans had first to be derivatized at this terminus, in this casewith 2-AP, after which the reaction worked well.

C. Other Derivative-Related Methods

A method that allows carbohydrates and other compounds to beexamined without a matrix involves formation of a photo-cleavable derivative that yields ions on irradiation with the laserpulse. The derivative consisted of several parts: a tether to capturethe target molecule, a linker and the photocleavable moiety. Thecarbohydrates, with a propargyl group (25) at the reducingterminus were captured with an azide group from the reagent togive 26. When exposed to the laser beam, the nitrobenzyl groupwas cleaved to leave a negative charge on the derivative, whichallowed detection by mass spectrometry. The negative modewaschosen to reduce fragmentation and to allow the method to beused quantitatively (Maki & Ishida, 2007).

VIII. GLYCAN ARRAYS

Glycan arrays are becoming common for glycan identificationand protein binding studies, and MALDI MS can play a role intheir construction. Thus, in a method developed by de Boer et al.(2007), glycans released from glycoproteins or glycolipids,both from natural sources or synthetic material, were labeledby reductive amination with common fluorescent labels such as2-AB or 2-aminobenzoic acid (2-AA, 1/57) purified by HPLCand characterized by MALDI-TOF or MALDI-TOF/TOF MSfromDHBorATT. The purified glycoconjugates were covalentlyimmobilized on commercial epoxide-activated glass slides viathe secondary amine group that linked the glycanmoiety with thefluorescent tag. Confirmation of the linkagewas also provided byMALDI-TOF MS. The resulting microarray provided informa-tive binding fingerprints for the lectin concanavalin A as well as14 monoclonal antibodies. Tseng et al. (2008) have built glycanarrays on aluminium-coated glass slides and found them to bemore sensitive than the transparent glass slides employed inprotein-binding analysis.

Microarrays have also been prepared from neoglycolipidsby oxime ligation to amodified phosphatidylethanolamine (4/49)with products characterized byMALDI-TOF analysis (Liu et al.,2007c). Microarrays were prepared with PVDF membranes orcoated glass slides and the oxime link from the carbohydrates tothe lipid did not appear to inhibit lectin binding, probably becauseNMRevidence suggested that rearrangement of the oxime link togive a closed ring structure had occurred. Oxime formation wasalso used by Seo et al. (2007b) for coupling aminophenylthiols tocarbohydrates so that they could be immobilized onto goldsurfaces.

Zhi et al. (2008) have also constructed glycan arrays on goldsurfaces using triethylene glycol-terminated C17 alkane thiol asthe linker. Aminated sugars were attached by succinimide esterchemistry whereas underivatized glycans were attached usinghydrazide chemistry. Ban and Mrksich (2008) have prepared a6� 4 array containing the disaccharides Gal–Gal, Glc–Gal,Gal–Glc, Glc–Glc, Gal–GlcNAc, and Glc–GlcNAc in all fourlinkages (b1! 2, b1! 3, b1! 4, and b1! 6), again on a goldtarget. The reaction time was 4 hr and the investigators used it toprofile the substrate specificity of bovine b-1,4-galactosyltrans-ferase I.

A microarray containing 144 natural and syntheticN-glycans has been prepared and used to screen for anti-glycanantibodies from patients infected with Schistosoma mansoni

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(de Boer et al., 2008). The glycans were first converted intotheir 2-AA or 2-AB derivatives and these were coupled toan epoxy-activated gold chip in fmol amounts, covalentbinding was between the epoxide and secondary amine of thederivative. MALDI-TOF analysis was used to analyze thederivatives but surface plasmon resonance was used to monitorthe array.

A review of glycoarrays for qualitative and quantitativeanalysis of carbohydrate binding has been published (Laurent,Voglmeir, & Flitsch, 2008a) and Konig (2008) has discussedthe use of bioaffinity target plates in general. Mrksich (2008)has reviewed recent work on mass spectrometry, particularlyMALDI, as a method for developing and characterizing a broadrange of chemical reactions of molecules attached to self-assembled monolayers of alkanethiolates on gold, a techniquetermed SAMDI-TOF mass spectrometry.

IX. QUANTIFICATION

Several studies on quantitation have been reported. Costello,Contado-Miller, and Cipollo (2007), during the development ofan LC/MSmethod for analysis of permethylated oligosaccharidealditols, observed that by permethylating carbohydrates, theybecome nearly chemically equivalent as seen the mass spec-trometer allowing semi-quantitative data to be derived from peakabundance measurements. MALDI-TOF analysis of suchglycans was used to measure the recovery of N-glycans releasedfrom decorin, a complex molecule from which a GAG chainneeds to be removed before N-glycan release. Using apermethylated malto-oligosaccharide as an internal standard ayield of approximately 70% based on an average of 75% siteoccupancy and a composite average molecular weight glycan of2,000Da was measured.

A recently developed technique that allows relative glycanquantification between two samples involves isotopic labeling ofone sample with methyl iodide and the other with [13C]-methyliodide using standard permethylation conditions. The glycanswere mixed in known proportions and the mixtures analyzed byMALDI and electrospray ionization-ion cyclotron resonance-MS (ESI-ICR-MS). The results indicated that the method wascapable of providing relative quantitative data with a dynamicrange of at least two orders of magnitude and adequate linearityand reproducibilitywith a coefficient of variation averaging 13%.The method was used to analyze N-linked glycan released froma1-acid glycoprotein, human transferrin, and bovine fetuin. Themeasured 12C/13C ratios frommixtures of glycans permethylatedwith either 12CH3I or

13CH3I were consistent with the theoreticalvalues (Alvarez-Manilla et al., 2007).

Seipert et al. (2008b) have usedmaltoheptaose reduced withsodium borodeuteride to add three mass units to its mass, toquantify fructooligosaccharides (inulin) with a MALDI-FT-ICRinstrument. Measurements at a single mass were sufficient toquantify the amount of inulin when the ratio of the constituentoligosaccharides was constant but, when these varied aswhen theinulinwas consumed byBifidobacterium longum, thesemeasure-ments had to be weighted. It was noted that the highest massdetected by the mass spectrometer was slightly different fromthat recorded by high-performance anion exchange chromatog-raphy-pulsed amperometric detection (HPAEC-PAD) due to themaximum sensitivity of the mass spectrometer being in the m/z

250–5,000 range. Other quantitative studies are included inthe sections below on specific carbohydrate types.

X. FRAGMENTATION

A. Post-Source Decay (PSD)

Cross-ring fragments have been observed as the main ions in thePSD spectra of acetylated monosaccharides with spectra thatwere similar to those obtained by electron impact (EI) eventhough the molecular ions ([MþNa]þ (even electron) fromMALDI and [MþNaþ] (odd electron) from EI) differed in theirelectronic configuration (Sato et al., 2007).

PSD from harmane of [MþCl]� ions from both anomers ofglucose disaccharides in 1! 3-, 1! 4-, and 1! 6-linkage hasgiven characteristic fragmentation patterns that enable thelinkages to be distinguished (Guan & Cole, 2007). Thus, theappearance of product ions atm/z 161, 179, 263, and 281with theabsence of m/z 251 appeared to be characteristic of the 1! 4-linked disaccharides. Observation of product ions at m/z 179,221, 251, and 281, with the absence of m/z 263, appeared to becharacteristic of the 1! 6-linked disaccharides, while observa-tion of B1 and C1 ions at m/z 161 and 179, together with a weakcross-ring cleavage ion at m/z 221, appeared to characterize1! 3-linked disaccharides. Anomeric differentiation was alsopossible in some cases. a-and b-1! 4-linked glucopyranosyldisaccharides gave a ratio of m/z 263:281 that was larger thanunity for the b-isomer or smaller than unity for the a-isomer. The1! 6-linked anomers could be readily differentiated by theabundance ratio of m/z 251:281 which was larger than unity forthe b-isomer but smaller for the other isomer. For the 1! 3glycosyl linkages, the relative abundances of m/z 161:179 wereconsistently lower for the a-isomer than for the b-anomer.

PSD fragmentation of a series of disaccharides, as propylglycosides, containing glucose, galactose and mannose attachedto glucose in different linkages together with some naturaldisaccharides (maltose, etc.), followed by linear discriminantanalysis, allowed both the linkage and residue type to bedetermined. A two-step method gave the best results: first, thecompounds were separated on the basis of linkage and thenresidue type in order to overcome problems associated with thelow abundance of fragment ions associated with glycans with1! 3-linkages (Zhang et al., 2008b).

The four halogen anions, acetate and nitrate have beenevaluated for their ability to yield negative ions and structurallyinformative PSD spectra from di-, tri- and tetra-saccharides andcyclodextrins (Guan & Cole, 2008). Fluoride and acetate werefound not to form adducts whereas bromide, iodide and nitrateyielded only the anions in their PSD spectra leaving chloride asthemost useful adduct. This is in contrast to recent work from ourlaboratory (unpublished) that has shown that nitrate gives equallyuseful fragmentation spectra but without the disadvantage of thepresence of two abundant stable isotopes. In thework reported byGuan and Cole, the linkage between glucose residues in glucosedimers could be determined by the relative abundances of certainions as listed in Table 1.

Negative ion mode has also been used to study the PSDfragmentation of a series of six glycosyl esters of nucleosidepyrophosphates and to verify the presence of one of thesecompounds in Saccharomyces cerevisiae (Heinrich et al., 2008).



3-Hydroxypicolinic acid and CHCA proved to be the bestmatrices; DHB and sinapinic acid gave poor results.

B. Collision-Induced Dissociation (CID)

1. High-Energy Collisions

A comparison of low and high-energy spectra of flavonoidshas shown considerable differences between these two energyregimes. Although high-energy spectra of small molecules gavemany structurally significant ions, only a few ions were producedfrom the larger molecules. Low energy spectra were obtained byESI on a Q-TOF instrument and byMALDI on a QIT instrumentand were virtually identical whereas high-energy spectra wererecorded with aMALDI-R-TOF instrument (March et al., 2007).High-energy fragmentation of glucosylated phytoalexins (27)from Chinese cabbage have also been reported (Bekesova et al.,2007) with a combination of MALDI-TOF and ESI-ion trapmass spectrometers. Much fragmentation involved cross-ringcleavages of the glucose molecule.

A comparison of several MS techniques for the structuralanalysis of two dammarane-type triterpenoid saponins (28)isolated fromB.monnieri has been reported by Zehl et al. (2007).

Low energy CID experiments were performed by ESI-ion trap(IT)-, atmospheric pressure MALDI (AP-MALDI)-IT-MS andMALDI-IT/reflectron time-of-flight (R-TOF) MS whereas high-energy collisions were studied with a MALDI-TOF/R-TOFinstrument. All fragmentation techniques clearly yielded thesequence and branching of the glycan moiety as well as themolecular mass of the intact aglycon. Cross-ring cleavages of thebranching sugar that were mainly observed in low-energy CIDgave some information on the sugar linkages, whereas high-energy CID yielded additional diagnostic fragment ions from theaglycon moiety. However, none of the MS techniques providedthe identification of those saponins that differed only by theiraglycon moiety (i.e., jujubogenin or pseudojujubogenin).

C. Photofragmentation

Photofragmentation with a 157 nm laser in an ion trap instrumenthas been shown,with ovalbumin glycans, to producemanyusefulcross-ring fragments (positive ion) not observable by CID(Devakumar et al., 2008). However, for complex mixtures, theauthors noted that ion production with ESI showed a bias againstthe larger ions and recommended MALDI as a more appropriateionization technique.

Ultraviolet photodissociation (UVPD) at 355 nm of fluo-rescently labeled glycans has been shown to produce comple-mentary fragmentation to CID. Unlike CID that produces

TABLE 1. Ions useful for determining the linkage of glucose dimers in negative ion PSD spectra

ecnesbAecneserPkniL 161:179Ratio

Relative ion abundance

1→6 − −162(179)

−120(221)

−90(251) − −60

(281) − −78(263) − <1 α252/281 <1

< β251/281

1→4 −180(161)

−162(179) − − −78

(263) −60

(281) −90

(251) − − >1 α263/281 <1< β263/281

1→3 −180(161)

−162(179)

−120(221) − − − − − − >1 α161/179 <1

< β161/179

1→2 − −162(179)

−120(221) − −78

(263) − − − −60(281) <1 α263/221 <1

< β263/221

1→1 − −162(179) − − − − − − − <1 −

Neutral losses are calculated from the mass of the [M�H]� ions.

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fragment ions containing the fluorophore by loss of fragmentsfrom the non-reducing terminus (Y-type ions), UVPD fragmen-tation results in fragments that have lost the fluorophore (A- andC-type ions). UVPD was found to give better isomeric differ-entiation of both lacto-N-fucopentaoses and lacto-N-difucohex-aoses, but in general, the combination of UVPD and CID wasfound to be most appropriate for complex branched oligosac-charides. Four fluorophores were examined and all yieldedsimilar MS/MS results; however, 6-AQ, (29), aminoacridone(AMAC, 1/58) and 7-aminomethylcoumarin (AMC, 1/47) hadmore efficient photon absorption and subsequent dissociationthan 2-AB. UVPD was also used for characterization of glycansreleased from ribonuclease B and derivatized with 6-AQ (Wilson& Brodbelt, 2008).

D. Infra-Red Multiphoton Dissociation (IRMPD)

Earlier reports of IRMPD fragmentation have been revised(Seipert et al., 2008a). Thus, in contrast to previously reportedaccounts that IRMPD gives only glycosidic bond cleavage, thefragmentation of singly protonated glycopeptides containing abasic amino acid residue has been shown to give almost exclusivepeptide backbone cleavage. Fragmentation of the doublyprotonated glycopeptides exhibited ions similar to thosepreviously reported; however, when the same glycopeptide wassodium coordinated, a previously inaccessible series of glycanfragments was observed. Molecular modeling suggested thatdifferences in the site of protonation and metal ion coordinationmight direct the glycopeptide ion fragmentation.

XI. ION MOBILITY MASS SPECTROMETRY (IMS)

The recent availability of commercial ion mobility massspectrometers and the popularity of several home-built deviceshave recently seen several applications in the carbohydrate field.Fenn and McLean (2008b) have demonstrated separations ofseveral compound classes such as carbohydrates, lipids, peptidesand nucleotides, each group of compounds falling into a separatebandwhenmobility is plotted againstm/z. Separation ofN-linkedcarbohydrates from peptides ionized by MALDI followingincubation of ribonuclease B with trypsin and protein N-glycosidase F (PNGase F) was also demonstrated. Boronic acidderivatization was later employed to improve confidence in thecarbohydrate characterization (Fenn & McLean, 2008a).

Olivova et al. (2008) have used a combination of MALDIand ESI-ion mobility to characterize the N-glycosylation sitesand site heterogeniety of a monoclonal antibody. In the IMSexperiment using the Waters Synapt instrument, HPLC-sepa-rated peptides and glycopeptides were fragmented in the trap cell(before the IMS cell) at low energy to fragment only the glycans.After mobility separation the peptides were fragmented in thetransfer cell at high energy, Confirmation of the glycan profile

was obtained by MALDI in a separate experiment after releasewith PNGase F.

XII. COMPUTER ANALYSIS OF SPECTRA

One of the problems that has been identified in glycomics isthe lack of comprehensive databases and software for glycanidentification. The review period has seen many attempts toovercome these problems and some useful software has beendeveloped. However, not all programs are capable of completeidentification of glycan structures and the predicted structuresshould only be taken as a guide.

A. Software for Interpreting Spectra

An improved version of the program ‘‘Cartoonist’’ whichannotates peaks in spectra of glycan mixtures has been reported(Morris et al., 2007). The software calculates all possiblestructures based on mass and then picks the most probable basedon the known biology of the target organism. Unfortunately, theprogram does not accommodate other information and, thus,the displayed structures are only proposals and the possibilitythat each mass peak might contain isomeric structures is notconsidered.

Another program, ‘‘Peptoonist,’’ automatically identifiesthe glycans present at each N-glycosylation site of a protein. Theinput is a series of both MS and MS/MS spectra obtained fromenzymatically digested glycoproteins. The program usesMS/MSto identify glycosylated peptides and MS to obtain monosac-charide compositions of the N-glycans present on each of thesepeptides. Analysis takes account of biochemical data and datagenerated by ‘‘Cartoonist.’’ The program was claimed to doublethe number of glycopeptide identifications, compared withmanual analysis and also to find several possible errors inthe hand annotation. In addition, the program automaticallyidentifiedmost of the sameglycan isomers as the expert annotator(Goldberg et al., 2007).

An algorithm that limits possibilities for N-linked glycansby taking account of the species from which the samples arederived is ‘‘GlycoPep DB’’ (http://hexose.chem.ku.edu/sug-ar.php). This application takes glycopeptide masses and assignsglycan compositions in terms of isobaric monosaccharidecompositions.Biologically implausible compositions are exclud-ed by knowledge of the sample origin, which, at the time ofpublication, included serum/plasma, pituitary hormones, andhuman immunodefficiency virus (HIV) envelope glycoproteins.The algorithm was claimed to be considerably faster thancomparable algorithms such as GlycoMod (http://www.expasy.org/tools/glycomod; Go et al., 2007).

‘‘GlyDB,’’ is a program for glycan structure annotation ofN-linked glycopeptides in low-energy tandem mass spectra ofglycopeptides derived from proteolysis of glycoproteins and useslow-energy collision-induced dissociation of N-linked glyco-peptides in which glycosidic bonds are preferentially cleaved. Atheoretical glycan structure database derived from biosyntheticrules for N-linked glycans was constructed in which the glycansare represented by linear sequences. The commonly used peptideidentification program, Sequest, was then utilized to assignstructures. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrated that the GlyDBapproach could be a useful tool for annotation of glycan



structures and for selection of a limited number of potentialglycan structure candidates for targeted validation (Ren et al.,2007).

A useful software tool called ‘‘GlycoWorkbench’’ has beendeveloped byEUROCarbDBwith the aim of considerably aidingthe interpretation of fragmentation spectra (Ceroni et al., 2008).It consists of a subroutine called ‘‘GlycanBuilder’’ (Ceroni, Dell,& Haslam, 2007) that enables the user to assemble a glycanstructure in one of several symbolic formats and then use the in-built fragmentation engine to calculate all possible fragmentswithin user-specified limits. The software can then matchcalculated fragments with ions in a spectrum and present theresults as an annotated spectrum. It has recently been extended tocover glycosaminoglycans (Tissot et al., 2008). The software isfreely available at http://www.eurocarbdb.org/.

The web-based service called ‘‘Glyco-peakfinder’’(www.eurocarbdb.org/applications/ms-tools; Maass et al.,2007) assigns compositions to all ions in a spectrum, includingfragment andmultiply charged ions. It can also handle themassesof derivatized glycans, both persubstituted and those withreducing-terminal modifications. Another mathematical methodfor generating glycan fragments from any N-linked glycan andfor calculating their masses has been developed by Meitei andBanerjee (2007). The article introduces the concept of positionalnomenclature, gives a systematic representation of glycanstructure of any size, and develops a method for theoreticallygenerating all possible first and second-generation fragmentsresulting from glycosidic and cross ring cleavages.

Another initiative arising from the EUROCarbDB project(http://www.eurocarbdb.org) is the development of a unifyingencoding sequence format for carbohydrates (Herget et al.,2008a). The encoding capabilities of all existing carbohydratesequence formats and the content of publically availablestructure databases were examined but none were found thatwere capable of coping with the full complexity to be expectedfor experimentally derived structural carbohydrate sequence dataacross all taxonomic sources. Consequently, an encoding schemefor complex carbohydrates, named ‘‘GlycoCT’’ was developedto overcome these difficulties. This new format is based on aconnection table approach, instead of a linear encoding scheme,to describe the carbohydrate sequences, with a controlledvocabulary to name monosaccharides, adopting IUPAC rules togenerate a consistent, machine-readable nomenclature. Theformat uses a block concept to describe frequently occurringspecial features of carbohydrate sequences such as repeatingunits. It exists in two variants, a condensed form and a moreverbose XML syntax. The condensed form is suitable as a directprimary key for database applications, which rely on uniqueidentifiers. Among other sequence formats is an XML descrip-tion of carbohydrate structures (Kikuchi, 2008b).

Another new program, called ‘‘GlycoMiner,’’ operates ontandem (MS/MS) spectra obtained by LC/MS and identifies N-glycopeptides from spectra obtained on various instruments(Ozohanics et al., 2008). The algorithm works similarly to ahuman expert; evaluates the low mass oxonium ions; deducesoligosaccharide losses from the protonated molecule; andidentifies the mass of the peptide residue. In a test of 3,132 MS/MS spectra, 338 were found to correspond to glycopeptides;identification by GlycoMiner showed a 0.1% false positive and0.1% false negative rate. From these it was possible to identify196 glycan structures manually; GlycoMiner correctly identified

all of these, with no false positives. The rest were low qualityspectra, not suitable for structure assignment. The software canbe downloaded from http://www.chemres.hu/ms/glycominer.

Another recently-developedweb utility (Sheng et al., 2008),available at http://ggdb.informatics.indiana.edu:8080/glycan-view, can be used to predict 1! 4 and 1! 6 linkages fromhigh-energy MALDI spectra recorded with a TOF/TOF instru-ment. Hamby and Hirst (2008) have published details of aprogram that can be used to predict glycosylation sites inglycoproteins. It can be found at http://comp.chem.nottingha-m.ac.uk/glyco/. The software was claimed to be better thancurrent glycosylation predictors and in tests, predicted glyco-sylation sites with an accuracy of 90.8% for serine (Ser) sites,92.0% for threonine (Thr) sites and 92.8% for asparagine (Asn)sites. There is also aweb-based tool named Structural GlycomicsCALculations (SGCAL) that is capable of building 3D structuresfrom sequence information (Fukui, 2008).

A statistical analysis has been performed of the bacterialcarbohydrate structure database presenting details such as thedistribution of carbohydrate sequences in various taxonomicclasses, the size distribution of carbohydrate sequence units, themost abundant monosaccharides in bacteria, the most abundantunique monosaccharides, branching and linkage (Herget et al.,2008b).

Because of the absence of a consensus sequence forO-linked glycosylation in glycoproteins, O-linked glycosylationsites are difficult to predict. A neural network prediction method,NetOGlyc3.1 (www.cbs.dtu.dk/services/netoglyc) has beendeveloped in an attempt to rectify this deficiency but itspredictions appear to fall short of ideal after a study found thatit only identified two of the six sites found by MALDI-TOFin a 20-residue segment of the insulin receptor b-chain. Onethreonine residue (T756) was incorrectly predicted to beglycosylated (Sparrow et al., 2007).

A tutorial that briefly describes several different bio-informatic methods for glycome research has been published(Aoki-Kinoshita, 2008).

B. Databases

Since the demise of the Complex Carbohydrate StructureDatabase (CarbBank) in 1997, several other partially overlappingdatabases have been developed but their different encodingschemes make it virtually impossible to obtain an overview ofall deposited structures. A new database, ‘‘GlycomeDB’’ hasimported structures from the sevenmajor databases together withtaxonomic annotations and references. The database containsmore than 100,000 datasets representing over 33,000 uniquesequences encoded in GlycoCT. Detailed installation instruc-tions can be found on the download web pages (http://www.glycome-db.org/downloads/; Ranzinger et al., 2008). Someother useful databases from Japan that have been described in thereview period are ‘‘KEGGGLYCAN,’’ containing 11,000 glycanstructures (http://www.genome.jp/kegg/glycan/; Hashimoto &Kanehisa, 2008), ‘‘GlycoEpitope,’’ a database of carbohydrateepitopes and antibodies (http://www.glyco.is.ritsumei.ac.jp/epitope/; Kawasaki, Nakao,&Tominaga, 2008), the ‘‘GALAXY’’database containing HPLC data for about 500 N-linked glycans(2-AP derivatives) (http://www.glycoanalysis.info/ENG/index.html; Kato & Takahashi, 2008), Glycoconjugate data bank(http://www.glycoconjugate.jp/;Miura&Nishimura, 2008a) and

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‘‘CabosDB,’’ a carbohydrate sequencing database (Kikuchi,2008a).

XIII. STUDIES ON SPECIFIC CARBOHYDRATETYPES

Most of the applications in this and the following sections aresummarized in tables with only details of some of the moresignificant methods described below. Applications of MALDIMS to the analysis of carbohydrates found in plant cell wallsand some animal tissues are listed in Table 2. Applications tocarbohydrates found in lower organisms are in Table 3. Manyof these compounds are too large for direct analysis by MALDIand need to be depolymerized enzymatically or by a suitablechemical method. Details for individual compounds are listed inthe tables.

A. Mono- and Oligosaccharides

A comparison of three reported methods for analyzing fructose,glucose, and sucrose in onions by HPLC coupled with anevaporative light-scattering detector and by MALDI-MS werefound to give differing results (Davis et al., 2007). The causewastraced to the extraction methods. Those based on a polar solventsuch as aqueous methanol gave more accurate results than thosebased on ethanol. The authors caution against interpreting someprevious work and tabulate 29 different studies.

A study of small oligosaccharides as their 2-AA derivativesusing normal-phase HPLC coupled to a 4,700 TOT/TOF massspectrometer (NP-HPLC-MALDI-TOF/TOF-MS/MS, or in theformat now used by some journals: NPHPLCMALDITOF-TOFMSMS) for high energy fragmentation, has enabled detailsof the structures of the oligomers to be determined (Maslenet al., 2007). Effluent from the HPLC column was spotted ontothe MALDI target with a Probot robot. The MS/MS spectracontained a wealth of cross-ring and ‘‘elimination’’ ions andthe technique allowed isomeric oligosaccharides produced bydifferent endo-xylanases with different substrate specificities tobe examined.

B. Polysaccharides

A comparative study of MALDI-TOF (linear mode, DHB),capillary electrophoresis (CE, 4-aminobenzonitrile derivatives)and HPAEC-PAD, (8-aminonaphthalene-1,3,6-trisulfonic acid(ANTS, 4/34) derivatives) has shown thatMALDI-TOF is able todetect higher molecular weight oligomers frommaltodextrin (upto DP-13) compared with the other two techniques (up to DP-9).MALDI-TOF, on the other hand gave a low response for theglucose monomer, unlike the other techniques. Partial hydrolysisof the carbohydrates by the derivatization technique wasproposed by the authors as a possible cause (Kazmaier et al.,1998). Two methods, solid-phase extraction and liquid chroma-tography for carbohydrate extraction and purification have beencompared with maltodextrins and commercial sugar syrup astest mixtures (Megherbi et al., 2008). Aminopropyl bondedphases were found to be the best for oligosaccharides withdegrees of polymerization greater than four. Both methods,combined with MALDI-TOF MS were then used to examinecarbohydrates, including those present in trace quantities, fromacacia honey.

Maltooligosaccharides derived from various beers havebeen used as mass calibrants by Clowers et al. (2008) on thepretext that they possess no memory effects as seen with manyothermass calibrants.Masses ([MþNa]þ ions) were observed tom/z 4,902 corresponding to DP30. Similar arrays of glycanscan be obtained from commercial preparations of dextran from,for example, Leuconostoc mesenteroides.

A method for the HPLC analysis of hexose oligomersfrom wheat (Triticum aestivum) that is claimed to be superiorto existing methods employing cyclodextrin phases has beenpublished (Robinson et al., 2007a). MALDI-TOF of permethy-lated glycans was used to characterize the mixture; permethy-lation was claimed to give superior results to analysis of the freeglycans. Hex19 (m/z 3,945) was the largest oligomer observed.MALDI imaging of cross- and longitudinal sections of wheat hasshown that a range of oligosaccharides up to Hex11 are presentand that these are located in the pith that is retained pre-dominantly around the inner stem wall (Robinson et al., 2007b).Burrell, Earnshaw, and Clench (2007) have investigated the useof MALDI imaging to monitor metabolites in wheat seeds.CHCA Produced signals from amino acids in positive ion modebut failed to detect carbohydrates. The matrix 9-aminoacridine,however, proved satisfactory allowing sucrose and glucose6-phosphate to be seen.

Several experimental variables such as the matrix (nor-harmane, DHB), sample preparation methods (mixture, sand-wich), inorganic salt addition (NaCl, KCl, NH4Cl), ion mode(positive, negative), linear and reflectron mode, etc. for theanalysis of highly methoxylated pectin have been evaluated byMonge et al. (2007). The best conditions were found to be the useof negative mode with nor-harmane as the matrix, a system notused before for the analysis of pectins. Its use avoided samplepre-treatment such as an enzymatic digestion or acid hydrolysis,and there was no need to add salts such as ammonium chloride.

A method for the quantitative sequencing of heterochitoo-ligosaccharides obtained by the enzymatic depolymerization ofchitosan has been developed byHaebel, Bahrke, and Peter (2007)using aMALDI-linear ion-trapMS. These compounds consist ofrandom chains of GlcN and GlcNAc but there is currently nomethod for quantitatively sequencing these compounds. In anyparticular mixture, any one mass can be represented by severalisobaric isomers leading to a complex analytical problem that sofar has not yielded to chromatographic techniques. Nevertheless,mass spectrometry has now provided an answer. Fragmentationof these compounds is mainly by B/Y, C/Z glycosidic cleavagesbut by tagging the reducing terminus with 3-(acetylamino)-6-aminoacridine (AA-Ac, 2/25) which protonates easily, fragmentions were almost exclusively Y fragments. However, the extentof cleavage varied depending on the nature (GlcN or GlcNAc)of the adjacent sugar residues. This problem was overcomeby converting the GlcN residues to GlcN[2H3]Ac. This reactiongave essentially a linear polymer of GlcNAc residues but with adeuterium label at the site of the originalGlcN residue. Cleavagesbetween the different groups were now equivalent allowingquantitative sequencing by MS2. Beyond the Y6 ion, MS3 hadto be used to extract the sequence. Several mixtures withcompositions up to GlcN4GlcNAc5 were fully analyzed by themethod.

A method for comparing arabinoxylans from spelt derivedfrom two plant species (wheat and oats) involves digestion withxylanase and derivatization with 13C6-(oats) and

12C6-(wheat)



TABLE 2. Use of MALDI MS for examination of carbohydrate polymers from plants

Species Carbohydrate Methods1 Notes Reference

Almond Xylo-oligosaccharides

TFA, TOF (DHB), NMR

Xylo-oligosaccharides isolated by autohydrolysis

are immunostimulatory

(Nabarlatz, et al., 2007)

Apple Pectin HCl, TFA, TOF (DHB), ESI, NMR

Link between homo- or xylogalacturonan and rhamnogalacturonan

(Coenen, et al., 2007)

Arabidopsisirregular xylem 8

mutant

Glucuronoxylan, homogalacturonan

Endoxylanase, endogalactanase, TOF

Deficiencies in xylan polymers affect secondary

cell wall integrity

(Persson, et al., 2007)

Arabidopsisthaliana Xylogalacturonan Xylogalacturonan

hydrolase, TOF (DHB)

Demonstration of xylogalacturonan in cell walls of various tissues

(Zandleven, et al., 2007)

Arabidopsisthaliana Glucuronoxylan β-Endoxylanase, TOF

(DHB)

PARVUS Gene in cells undergoing secondary

wall thickening essential for glucuronoxylan

biosynthesis

(Lee, et al., 2007c)

Arabidopsisthaliana Glucuronoxylan

Trichoderma virideendoxylanase, TOF

(DHB/HIQ)

Genetics of biosynthesis. Reducing end structure

evolutionarily conserved in plants

(Peña, et al., 2007)

Arabidopsisthaliana Xyloglucan

XyG-specific endo-β -1,4-glucanase, R-TOF

(DHB)

Disrupting two Xyl-transferase genes shown to

give plants deficient in xyloglucan

(Cavalier, et al., 2008)

Arabidopsisthaliana Xylan Endo-β -xylanase, TOF

(DHB), glycans (2-AA)

The irregular xylem9 mutant deficient in xylan

xylosyltransferase activity (Lee, et al.,

2007b)

Arabidopsisthaliana Xylan HAc, TOF/TOF (DHB)

Comparison of five mutants. New insight into

xylan synthesis

(Brown, et al., 2007)

Argania spinosa Pectin and xyloglucan

Endo polygalacturonase, TOF (DHB), GC/MS, GLC

Structural identification from fruit pulp cell wall

carbohydrate

(Aboughe-Angone, et al.,

2008)

Avena sativa (Oat) Fructans TOF (DHB), HPLC

Cereal fructans stabilize cellular membranes during dehydration.

(Hincha, et al., 2007)

Bletilla striata (Orchid) Polysaccharides TOF (DHB) Molecular weight

measurements (Cheng, et al.,

2008a) Castanea sativa(Sweet chestnut)

4-O-Methyl-glucuronoxylan

Water (100oC), TOF (DHB), NMR Structural determination (Barbat, et al.,

2008)

Citrullus lanatus(Watermelon) Xylogalacturonan

Endopoly galacturonase CAZY family 28 from Aspergillus niger, R-TOF (THAP), ESI,

MS/MS

Structural determination from cell-wall pectin.

Xyl residue on the GalA after hydrolysis site

(Mort, et al., 2008)

Cowpea starch Amylose, amylopectin

Enzymatic (various), TOF (DHB), HPAEC,

SEC

To study acetylation by Ac2O and vinyl acetate

(Huang, et al., 2007b)

Enteromorpha compressa (Seaweed)

Heteroglycans Xyloglycans

Endo-glucanase, amyloglucosidase, TOF (DHB), GLC, GC/MS,

HPLC

Structural determination of sulfated carbohydrates

(Chattopadhyay, et al., 2007b)

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TABLE 2. (Continued )

Fleurya aestuans Xyloglucan, (West Indian woodnettle)

rhamno-galacturonan

Endo-glucanase and endo-xylanase, TOF

(DHB), GLC Structural determination (Angone, et al.,

2008)

Gossypium hirsutum L.

Rhamno-galacturonan

Several enzymes, TOF (THAP), GLC, NMR

Structural determination of novel oligosaccharide

(Zheng & Mort, 2008)

Hordeum vulgare (Barley)

Polysaccharides from endosperm

cell walls

Water, NaOH extn. Licheninase, Q-TOF (DHB), Glycans (per-

Me) GC/MS

Structural determination from three Canadian

regions

(Lazaridou, et al., 2008)

Hordeum vulgare (Barley) β-Glucan TOF (s-DHB), NMR,

HPLC Structural determination (Ghotra, et al., 2008)

Lagenaria siceraria (Lau)

Methyl galacturonosyl-methoxyxylan

TFA, TOF (DHB), GC/MS, NMR, OR

Structural determination from stem

(Ghosh, et al., 2008b)

Laminariagurjanovae

Alginic acid, laminaran, fucoidan

Acidic, TOF (DHB, arabinosazone)

Analysis of polysaccharides and lipids from the brown seaweed

(Shevchenko, et al., 2007)

Linumusitatissimum L.

(Flax)

Fibre-specific (1-4)-galactan

Aspergillusaculeatus endo-

galactanase,R-TOF (DHB), SEC,

HPAEC, NMR

Structural determination (Gur’janov, et al., 2007)

Paulownia elongata/P.

fortunei hybrid

Acetylatedheteroxylan

TFA, TOF, TOF/TOF (DHB, CMBT), ESI,

MS/MS, NMR Structural determination (Gonçalves, et

al., 2008)

Phragmenthera capitata

Xyloglucan, rhamno-

galacturonan

Endo-glucanase and endo-xylanase,

TOF (THAP), GLC, NMR

Structural determination (Angone, et al., 2008)

Picea abies (Norway spruce) Non-structural TOF/TOF (DHB), ESI

Sucrose content affected by light and CO2

concentration

(Cabálková, et al., 2007)

Picea abies(Norway spruce)

O-Acetyl galacto-glucomannans

Water and HCl, R-TOF (DHB)

Kinetics of acid hydrolysis (Xu, et al., 2008)

Prosopis velutina

(Mesquite) Gum

α-Galactosidase, R-TOF (DHB), FT-IR,

GC/MS, GLC Structural determination (López-Franco,

et al., 2008)

Secale cereale (Rye) Fructans TOF (DHB), HPLC

Cereal fructans stabilize cellular membranes during dehydration.

(Hincha, et al., 2007)

Sesame meal Xylan Endo-β-1-4-xylanase,TOF, HPLC Structural determination (Chattopadhyay,

et al., 2007a)

Solanum tuberosum

(Potato) Methylated starch

α-Amylase and amyloglucosidase,

TOF (DHB), GC/MS

Substitution patterns in methylated potato starch. Structure and composition of fragments in enzymatic

digests

(Steeneken, et al., 2008)

Tamarindus indica

(Tamarind) Xyloglucan

Xyloglycanase from Phanerochaete chrysosporium,

TOF (DHB), HPLC

Study of substrate recognition by the

enzyme

(Ishida, et al., 2007)

Triticum spp (Wheat) Arabinoxylans

Endo-β-(1-4)-xylanase,TOF/TOF (DHB),

HPLC, glycans (2-AA derivs.)

NP-HPLC-MALDI-TOF/TOF-MS/MS

method. High energy fragmentation

(Maslen, et al., 2007)



aniline (Ridlova et al., 2008). Identification of the carbohydrateswas made by off-line LC-MALDI-TOF/TOF MS/MS withquantification by LC-ESI-MS. Xyloglycans from Vacciniummyrtillus (Bilberry) are among the most complex xyloglycansyet described. Hilz et al. (2007) have used several techniquesincluding HPAEC, MALDI-TOF, ESI-MS, and NMR andidentified over twenty building blocks including four that havenot been described before.

Murlikrishna and Subba Rao (2007) have reviewed workon cereal non-cellulosic polysaccharides with special referenceto the recently characterized finger millet arabinoxylans.

Fifty percent of the carbon in marine high molecular weightdissolved organic matter (HMWDOM) from the ocean surfaceconsists of acylated polysaccharides that are conserved acrossocean basins. However, acid hydrolysis of HMWDOM, followedby chromatographic analysis, recovers only 10–20% of the


Triticum spp (Wheat)

Arabinoxylo-oligosaccharides

Xylosidase, hemicellulolytic fungal enzyme,acetyl-xylan

esterase,TOF (DHB/TFA)

MALDI analysis of hydrolysis products.

Feruloylated and acetylated pentose

oligomers.

(Sørensen, et al., 2007)

Triticum spp (Wheat)

Hordeum vulgare (Barley)

Lignocellulose from straw

Cellulase (Trichoderma reesei), β-glucosidase,

(Aspergillus niger), TOF

Evaluation of water or acid impregnation followed by steam

explosion or hot water extraction as pretreatment

(Rosgaard, et al., 2007)

Vaccinium myrtillus(Bilberry)

Xyloglycan

Endo-glucanase, Polygalacturonase,

pectin methyl esterase, R-TOF (DHB),

HPAEC, ESI-MSn, CE-ESI-MS, NMR

One of the most complex xyloglycan structures yet described. More than 20

building blocks

(Hilz, et al., 2007)

Various (Wheat, oats, eucalyptus) Xylans TOF (DHB), NMR

To study the binding of xylans to bacterial

cellulose

(Kabel, et al., 2007)

Various, mainly commercial

varieties

Various polysaccharides

Xyloglucanase, cellobiohydrolase, β-D-

glucosidase, β-galactosidase, TOF

Structural determination of polysaccharides from

13 vegetables and 11 fruits

(Kato, 2008)

Wheat starch Arabinoxylans

“Celluclast 1.5 L” and “Ultraflow L”,

R-TOF (DHB), ESI, GC/MS

Characterization of oligosaccharides from industrial fermentation

residues

(Matamoros Fenández, et al.,

2007)

Wood pulp Xylans “Celluclast”, TOF (DHB)

Investigation of bioethanol production from pulp mill sludge

(Sjöde, et al., 2007)

Yellow pea Starch α-Amylase,

TOF (DHB), HP-SEC, HPAEC

To study the effects of acetylation.

(Huang, et al., 2007a)

Commercial Cellulose Hydrolysis, TOF/TOF, LC, NMR

Hydrolysis by amorphous carbon bearing SO3H,

COOH, and OH groups

(Suganuma, et al., 2008)

Commercial Cellulose (from filter paper)

Hot water and H2SO4 or phosphoric acid, TOF

To examine degree of cross-linking caused by water or acid treatment

(Chaiwat, et al., 2008)

Commercial Carboxymethyl-cellulose

Endoglucanase, TOF (DHB + (NH4)2SO4)

Correlation between chemical structure and rheological properties

(Enebro, et al., 2007)

Not stated Starch, pectic acid Hydrothermal, TOF Hydrothermal degradation

of polysaccharides in a semi-batch reactor

(Miyazawa, et al., 2008)

1Format (not all items present): Depolymerization method, MALDI method (matrix), compounds run (derivative), other methods.

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TABLE 3. Use of MALDI MS for examination of carbohydrate polymers from lower organisms

Species Carbohydrate Methods1 Notes Reference

Alloiococcus otitidis Capsular polysaccharide

L-TOF (sinapinic), ESI, GC/MS, NMR

Structural determination and synthesis of protein

(BSA) conjugate

(Arar, et al., 2008)

Auricularia auricula-judae

(fungus) β-Glucan TOF (DHB), GLC,

GC/MS, NMR

Structural determination. Chain of (1→4)-linked D-

Glcp with Glcp side groups at O6.

(Ma, et al., 2008)

Bacillus cereus ATCC 10987

Secondary cell wall polysaccharide

TOF (DHB), GC/MS, NMR

Structural determination. Same HexNAc3

trisaccharide as B. anthracis.

(Leoff, et al., 2008)

Corynebacterium glutamicum Arabinogalactan R-TOF (DHB) Study of enzymology

involved in biosynthesis (Meniche, et al.,

2008)

Equisetum arvense(horsetail) Cell walls TOF, Glycans (per-

Me)

Mixed-linkage D-glucan not unique to plants of

Poales order.

(Sørensen, et al., 2008)

Erwiniachrysanthemi Galactan TOF (2,3-DHB) Investigation of galactan

utilization (Delangle, et al.,

2007)

Ganoderma lucidum(fungus) β-Glucan

TOF/TOF (DHB, THAP), Glycans

(free and per-Me), GC/MS

Structural determination (Hung, et al., 2008)

Geobacillusstearothermophilus

PV72/p2 Secondary cell wall

polymer

L-TOF (DHB), ESI-Q-TOF, NMR

Structural determination. Pyruvic acid acetal linked

to α-D-ManpNAc

(Petersen, et al., 2008)

Gluconacetobacter hansenii PJK GlcA oligomers TOF (CHCA)

Structure and physical properties from beer fermentation broth

(Khan & Park, 2008)

Gracilaria corticata Water-extracted polysaccharides

TOF, GC/MS, NMR

Chemical characterization and anti herpes simplex

virus activity

(Chattopadhyay, et al., 2008)

Hirudo medicinalis(leech)

Oligosaccharides binding to Lan3-2

antibody

R-TOF (DHB), glycans (Per-Me),

ESI, GC/MS, NMR Structural determination (Huang, et al.,

2008)

Lactobacillus reuteri α-D-Glucan (EPS35-5)

TOF (DHB) GC/MS, NMR

Structural analysis of α-D-glucan (EPS35-5) from the L. reuteri strain 35-5

glucansucrase GTFA

(van Leeuwen, et al., 2008c)

Lactobacillus reuteri α-D-Glucan (EPS180)

TOF (DHB) GC/MS, NMR

Structural analysis of α-D-glucan (EPS180) from the L. reuteri strain 180 glucansucrase GTF180

(van Leeuwen, et al., 2008b)

Lactobacillus reuteri Strain 180 α-D-Glucan TOF (DHB)

GC/MS, NMR

Generation of α-(1→4)linkages in native glycan

by triple mutant

(van Leeuwen, et al., 2008a)

Mesorhizobium loti Periplasmic glucans TOF (DHB), NMR

Structural determination. Glycerylphosphoryl and

succinyl residues

(Kawaharada, et al., 2008)

Mycobacteriumbovis

Extracellular polycaccharide

TOF, Glycans (per-Me), GC/MS,

NMR

Structural determination. α-glucan less accessible to pullulanase (debranching

(Dinadayala, et al., 2008)

enzyme), than glycogen



carbon as neutral, amino and acidic sugars. Panagiotopoulos,Repeta, and Johnson (2007) have used acid hydrolysis followedby Agþ and Pb2þ cation exchange chromatography to separateHMWDOMhydrolysis products and characterized themwith 1-Dand 2-D NMR and MALDI-TOF MS. 3-O-Methylglucose (30),3-O-methylrhamnose (31), 2-O-methylrhamnose (32), and 2-O-methylfucose (33) were identified in addition to neutral sugarsidentified in earlier studies. Some unidentified 3-deoxysugarswere also present. The authors found that most HMWDOMcarbohydrate is not depolymerized by acid hydrolysis and that thenon-hydrolyzable component includes 6-deoxysugars.


Mycobacteriumkansassii

Arabinomannan, phosphorylated

mannan

TOF (DHB), Glycans, NMR Structural identification (Maes, et al.,

2007)

Mycobacteriumsmegmatis D-Arabinans

TOF/TOF (DHB), Glycans (Per-Ac,

per-Me)

Characterization of arabinouranosyl-

transferase

(Zhang, et al., 2007c)

Mycobacteriumsmegmatis Synthetic galactan TOF, TOF/TOF

(DHB)

Transfer of the first Arafresidue to galactan is essential for viability

(Shi, et al., 2008)

Mycobacteriumtuberculosis

Capsular glucan and glycogen

R-TOF (DHB), GLC, GC/MS,

NMR

Structural determination, biosynthesis and impact on persistence in mice

(Sambou, et al., 2008)


Mycolyl arabinogalactan

R-TOF, per-Ac sugars, GC/MS,

NMR

Structural determination and location of succinyl

residues

(Bhamidi, et al., 2008)

Physcomitrellapatens (moss) and

Marchantia polymorpha(liverwort)

Xyloglucan TOF, ESI, NMR

Moss and liverwort xyloglucans contain GalA Structurally distinct from those synth. by hornworts

and vascular plants

(Peña, et al., 2008)

Pseudomonas aeruginosa

Linearosmoregulated

periplasmic glucans

TOF (DHB), GC/MS Genetics of biosynthesis (Lequette, et al.,

2007)

Ralstoniasolanacearum

acetylated α-cyclo-sophorotridecaose TOF (DHB), NMR Structural determination (Cho, et al.,

2008) Sinorhizobium

meliloti-Medicago truncatula Symbiots

Succinoglycans TOF (DHB), FAB, NMR

Strain-ecotype specificity correlated to

succinoglycan structure

(Simsek, et al., 2007)

Volvariellabombycina (fungus)

Heteroglycan extracted with hot

water

R-TOF (DHB), GC/MS, NMR Structural determination (Das, et al.,

2008)

Volvariella diplasia (fungus)

Glucan (water soluble)

TOF (DHB), GC/MS, ORD,

NMR Structural determination (Ghosh, et al.,

2008a) 1Format (not all items present): MALDI method (matrix), compounds run (derivative), other methods.

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C. Cyclodextrins and Related Compounds

MALDI-TOFMS has been used to characterize microbial cyclicb-(1–3),(1–6)-glucans (34) from Bradyrhizobium japonicum,used for the chiral separation of flavones (35–39) and (þ)- and(�)-catechin (40) (Kwon, Park, & Jun, 2007a; Kwon et al.,2007b). Yamane et al. (2008) have tried, but failed to observea complex between b-cyclodextrin and phenobarbital (41).With DHB as the matrix, the [MþNa]þ ion from b-cyclodextrinwas observed and from nor-harmane only the deprotonatedmolecule ([M�H]�) was obtained. A spectrum was finallyobtained by ESI.



D. Carbohydrates From Milk

Experimental details of a method for examination of milkoligosaccharides have been published (Urashima, 2008). Thecarbohydrate fractions were separated frommilk or colostrum byextraction with chloroform/methanol and the oligosaccharideswere purified by gel filtration, ion exchange and HPLC.Each oligosaccharide was characterized by 1H, 13C, 1H–13CHSQC NMR spectra and MALDI-TOF MS. The article alsocontains a list of 25 mammals whose milk has been analyzed.

A method for quantifying oligosaccharides in human milkhas been developed to measure the rate of consumption ofthese compounds by bacteria such as Bifidobacterium longum.A deuterated internal standard was found to provide betterprecision than an external standard.Milk sampleswere incubatedwith and without the bacterium; the samples without bacteriawere reduced with NaB2H4 to introduce one deuterium atomwhereas the fermented samples were reduced with unlabeledreagent. Samples were thenmixed and examined byMALDI-FT-ICRMS from DHB. The rate of consumption of different sugarscould be measured in the same sample by the 1H2/

2H2,13C ratios

and, of the three bacteria tested, Bifidobacterium longum wasfound to be the most efficient at digesting the sugars (Ninonuevoet al., 2007). High-molecular weight fractions obtained via gelpermeation chromatography of neutral human milk oligosac-charides have been investigated as per-methyl derivatives byMALDI-FT-ICR mass spectrometry from DHB and CMBT andshown to contain more complex neutral oligosaccharides thanhave been described previously. The oligosaccharides were oftenhighly fucosylated and their poly-N-acetyllactosamine coreswere substituted with up to 10 fucose residues on anoligosaccharide containing 7 N-acetyllactosamine units. Thisreport was said to be the first describing the existence in humanmilk of this large range of highly fucosylated oligosaccharides,manyofwhich possess novel, potentially immunologically activestructures (Pfenninger et al., 2008).

E. Glycoproteins

Several reviews on the analysis of glycoproteins have beenpublished. These includemass spectrometry in glycomics (Peter-Katalinic, 2007; Zaia, 2008) and glycobiology (Rodrigues et al.,2007a), glycopeptide analysis (69 references; Dalpathado &Desaire, 2008), tandem mass spectrometry of glycopeptides,Pan et al. (2007; Wuhrer et al., 2007), application of massspectrometry to glycoprotein hormones such as human chorionicgonadotropin (CG) and luteinizing hormone (LH) (Kicman,Parkin, & Iles, 2007) integrated analytical strategies for glyco-and phospho-proteins (150 references; Temporini et al., 2008),

proteome research (Chen, 2008), glycosylation of insect cells(100 references); (Rendic, Wilson, & Paschinger, 2008),glycosylation analysis of glycoproteins by capillary electro-phoresis and its interfacing with mass spectrometry (Amon,Zamfir, & Rizzi, 2008), S-layer glycoproteins (Messner et al.,2008) and carbohydrate interactions relating to fertilization in thepig (Tofer-Petersen et al., 2008). This last review contains severalMALDI-TOF profiles of N-glycans extracted from variousreproductive tissues.

1. General Comments on the Analysis of IntactGlycoproteins

An extensive compilation of proteins from normal human livertissue has been published. Proteins were separated by 2D gelelectrophoresis and identified by MALDI-TOF MS. About halfof the identified compounds were glycoproteins (Zhou et al.,2007a). Glycosylation of larger glycoproteins were detected inMALDI-TOF spectra by peak broadening which can sometimesbe extensive as shown by the spectrum of the hepatitis C virusenvelope glycoprotein (Iacob et al., 2008a,b). Broad MALDIpeaks (m/z 23.4 kDa) with masses higher than that calculatedfrom the amino acid sequence, recorded in linear mode, haveindicated glycosylation in the fusion protein shFas from HeLacells (Li et al., 2007h). Smaller glycoproteins with multipleglycosylation sites also give broad unresolved peaks as shown bya recent study of human pituitary follicle-stimulating hormone(hFSH; Bousfield et al., 2007). However small glycoproteinswith only one occupied site can often give several peakscorresponding to specific glycoforms. Electrospray ionsources are frequently used in combination with instruments ofhigher resolution and in these cases it is easier to resolveglycoforms as illustrated by a recent study by Satterfield andWelch (2005).

Several matrices and sample preparation techniques havebeen examined with respect to their influence on the measuredmass of intact glycoproteins with different degrees of glyco-sylation (Gimenez et al., 2007). Glycoproteins examinedincluded human transferrin, bovine fetuin, bovine a1-acid-glycoprotein and recombinant human erythropoietin. Sinapinicacid (1/48) was shown to cause considerable fragmentation at anylaser intensity, suggesting that this ‘‘hot’’ matrix is unsuitable fora reliablemolecularmass determination of glycosylated proteins.Improved results were obtained with DHB and ATT; thesematrices produced less fragmentation and gave results compa-rablewith those masses reported in the literature. Ethanol provedto be a superior solvent than acetonitrile, particularly with ATT,where large leaf-shaped crystals were produced. Sensitivity wassimilar to that obtained with sinapinic acid.

2. N-Linked Glycans

General work on the structural determination of N-glycans hasbeen reviewed (Harvey, 2007), characterization of glycosylatedproteins by mass spectrometry has featured in a book chapter byHagglund and Larsen (2007) and work onN-linked glycans fromschistosomes has also been reviewed (Hokke et al., 2007).

a. Experiments with intact N-linked glycoproteins. MALDI-QIT-TOFmass spectrometry has helped in the development of anassay for the oligosaccharide transferase that attaches the lipid-

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linked oligosaccharides to the consensus N-linked site on theprotein. The assay involved enzymatic attachment of the glycanto a synthetic, fluorescently labeled peptide that was separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS–PAGE) and assayed by fluorescence. The method wasused to detect enzyme activity in the membrane fraction from ahyperthermophilic archaeon, Pyrococcus furiosus (Kohda et al.,2007). A method for detecting the presence of non-reducingGlcNAc residues on N-glycans of glycoproteins makes use of amutant b1,4-galactosyltransferase that is able to transfer reactive2-keto-galactose that provides a functional handle for developinga highly sensitive chemoenzymatic detection method (Boegge-man et al., 2007). The location of the transferred 2-keto-galactoseon immunoglobulin G (IgG) and ovalbumin glycoproteins wasconfirmed by MALDI-TOF after removal of the glycans withPNGase F.

b. Clean-up and extraction. Separating glycoproteins fromproteins is often difficult and has resulted in the developmentof several separation techniques. Thus, Alves et al. (2007)have developed a method involving protein solubilization andextraction using cobalt or nickel chelating magnetic beads for

examination of the integral membrane protein, the tachykininNK-1 receptor, a member of the G-protein coupled receptorfamily. The beads were added directly to the MALDI targetwith CHCA matrix. Glycosylation was confirmed by PNGaseF digestion. Sparbier et al. (2007) have used magnetic beadscontaining various lectins or boronic acids for extraction andfractionation of serum glycoproteins. Derived tryptic glycopep-tides were subsequently analyzed by LC-MALDI-MS/MS.

Shimaoka et al. (2007) have developed a resin containingaminooxy functions (N-[2-[2-(2-tert-butoxycarbonylaminooxy-acetylaminoethoxy)ethoxy]ethyl]2-methacrylamide, 42) thatcan form oxime bonds with the reducing ends of carbohydrates.Glycans from ribonuclease B were used as model compounds.Once bound, the glycans could be cleaned and then released bytrans-oximation in the presence of O-substituted aminooxyderivatives under weakly acid conditions. Using this method, theauthors recovered twenty-three N-glycans from human serumand showed a MALDI-TOF profile (Fig. 2) that was very similarto those obtained by other methods. In an extension to themethod, the reducing ends of the glycans of glycoproteins wereoxidized with galactose oxidase, bound to the resin and theproteins were released with PNGase F.

FIGURE 2. MALDI-TOF spectrum of benzyloxyamine-labeled N-glycans released from human serum

glycoproteins following desialylation. Inset: MALDI-TOF mass spectrum of digest mixture without

glycoblotting. Reprinted with permission from Shimaoka et al. (2007). Copyright 2007 John Wiley and

Sons, Inc.



A similar method termed ‘‘reverse glycoblotting’’ has beendeveloped by Kurogochi et al. (2007) for enrichment of trypticglycopeptides containing sialylated glycans. The sialic acidswere oxidized on the side chain with sodium periodate to give analdehydes at C7 which were reacted with Fischer type polymerreagents, such as aminooxy-functionalized polyacrylamide orhydrazide-containing polymers, to form stable oxime bonds.After separation by gel-filtration, the covalently captured glycanswere released by treatment with 3% TFA at 1008C for 1 hr todigest the a-glycoside bonds between the sialic acid and theadjacent galactose residues. The method was evaluated withglycans from human fetal-cord serum a-fetoprotein (AFP),bovine pancreatic fibrinogen, and recombinant human erythro-poietin (rHuEPO), expressed in Chinese hamster ovary (CHO)cells (See Tables 4 and 6).

The ability of boronic acids to bind glycans has been utilizedby Yeap, Tan, and Loh (2008) to extract glycoproteins fromglycoprotein-protein mixtures. 3-Aminophenylboronic acid(5/42) was coupled via an alkyl spacer chain and a silyl groupto detonation nanodiamonds and mixed with a mixture ofalbumin (non-glycosylated), ovalbumin and RNase B (glycosy-lated) and then separated by microcentrifugation. The extractedcomplex was mixed with the MALDI matrix (CHCA) andexamined by linear MALDI-TOF. Strong signals were obtainedfrom the glycoproteins but none were obtained from albumin.Magnetic nanoparticles bound to aminophenylboronic acid havealso been used to separate glycopeptides and glycoproteins (Zhouet al., 2008b).

Hydrazide chemistry has also been used to captureglycoproteins for protein identification but the method involvedglycan modification and was thus not useful for subsequentglycan profiling (Sun et al., 2007). The proteins and glycopro-teins were digested with trypsin and the glycans were oxidizedwith periodate. The resulting aldehyde groups allowed theoxidized glycopeptides to be captured by hydrazide chemistry ona solid support, after which the unbound peptides were washedaway. Release of the peptides for mass spectrometric analysiswas achieved with PNGase F. A feature of the procedure was thequenching of the periodate reaction with sodium sulfite allowingthe complete sequence of reactions to be completed in a singlevessel. The method was validated with well-characterizedglycoproteins such as invertase (yeast), a1-antitrypsin (human),conalbumin (chicken), ribonuclease B (bovine), and ovalbumin(chicken), and applied to microsomal fractions of the cisplatin-resistant ovarian cancer cell line IGROV-1/CP.

Another recent method for glycoprotein analysis makes useof the affinity of sialic acids for TiO2.Microcolumnswere packedinGELoader tips by first inserting a small plug of C8material thatwas stamped out of a 3M Empore C8 extraction disk using anHPLC syringe needle or similar device and placed at the end ofthe tip. TiO2 beads, suspended in acetonitrile, were packed on topof the C8 disk using a 1-mL disposable syringe. Tryptic peptidemixtures were diluted five times in loading buffer (1M glycolicacid in 80% acetonitrile, 2–5% TFA) and loaded onto the TiO2

microcolumn which was washed with 10 mL of loading bufferfollowed by 40 mLofwashing buffer (80%acetonitrile, 2%TFA).The sialic acid-containing glycopeptides were eluted using 20–40 mL of aqueous ammonia. A small aliquot of each of the eluateswas acidified with 100% formic acid, purified using a Poros R3reversed phasemicrocolumn, and analyzed byMALDI-TOFMS.The method was used to characterize the human plasma and

saliva sialiomes where 192 and 97 glycosylation sites, respec-tively, were identified (Larsen et al., 2007). Experimental detailsfor enrichment of glycopeptides by Sepharose CL4B or Super-ose-12 have also been published (Wada, 2008a).

c. Detection of glycan presence from mass differences. Thepresence of glycans attached to a protein is often detected bymass difference before and after deglycosylation or between thesequence and measured masses, and sometimes the nature of theglycans can also be deduced. Thus, for example, the measuredmass of 50,874Da,�126Da for human hyaluronidase-1 insteadof the calculatedmass of 47,368Da. arises from posttranslationalN-linked glycosylation. The amino acid sequence of hHyal-1contains three predicted N-glycosylation sites at Asn-99, Asn-216, and Asn-350; thus the estimated average N-glycan massis 1,169Da per site, in agreement with previously reportedbiantennary carbohydrates linked to proteins expressed inDrosophila cells (Chao, Muthukumar, & Herzberg, 2007). Thedifference between the calculated (66,553Da) and measuredmass (67,166Da) for the 16 heme cytochrome from Desulfovi-brio gigas measured by Santos-Silva et al. (2007) allowed theauthors to conclude that the protein probably contained threeGlcNAc residues. Miller et al. (2008) have measured the mass ofa flagellin mutant as 40,197 (�10) Da, which would correspondto the mass of flagellin with two deoxyhexose substituents eachwith a mass of 146Da. However, the location of the sugar wasnot determined. The molecular mass of the glycoprotein drugCerezyme1 decreased by an average of 1,347.9� 367.7Daafter N-glycosidase F treatment implying loss of approximately6–7 sugar residues (Kacher et al., 2008).

d. Site occupancy. Having established that a protein is glyco-sylated, the next task is to determine which asparagine of theconsensus N-linked amino acid sequences (Asn-Xxx-Ser(Thr) isoccupied and with what glycans. Normally this is done by trypticor other proteolytic digest of the protein to isolate each site to aspecific glycopeptidewhich can then either be examined directlyby mass spectrometry (LC-MS or MALDI) or the glycanscan be removed enzymatically or chemically and the individualconstituents examined independently. The mass differencebefore and after deglycosylation provides some information onthe glycan composition. If the glycans are removed with PNGaseF, an amidase, an aspartic acid is left at the site of glycanattachment, increasing the mass of the peptide by 1Da. Severalexamples of this technique have appeared in the review period.Thus, for example, Khoshnoodi et al. (2007) have identified 9 ofthe 10 potential glycosylation sites in nephrin by this technique.The method was augmented by observation of preferentialcleavage of the amide bond carboxyl-terminal to aspartic acidresidues in peptides where the charge was immobilized by anarginine residue. Of five potentialN-linked glycosylation sites ofthe mutant protein, glucocerebrosidase in Gaucher’s disease,three (Asn-59, 146, 270) were shown to be glycosylated, one(Asn-462) was not glycosylated but a relevant peptide containingthe fifth site, Asn-19, was not observed (Bleijlevens et al., 2008).Kario et al. (2008) have used the Asn to Asp conversion toshow that N-glycosylation on ribonuclease B does not impairproteosomal degradation by PNGase F. Other uses of theconversion include studies on the membrane protein CD9P-1(Andre et al., 2007a) and studies on polysialylation of the neuralcell adhesion molecule (NCAM; Galuska et al., 2008).

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TABLE 4. Use of MALDI MS for examination of N-glycans from specific glycoproteins

Glycoprotein Type Methods1 Notes Reference

Acidceramidase Human

PNGase F, R-, L-TOF (CHCA, sinapinic)

glycoproteins, glycopeptides

Characterization of enzyme. N-glycans confirmed by ESI

after release

(Schulze, et al., 2007)

α1-Acid glycoprotein Human Trypsin, TOF

(DHB/CHCA) Optimization of methods for

glycopeptide analysis (Zhang, et al.,

2008d)

α1-Acid glycoprotein Human

PNGase F, TOF/TOF (DHB), High-mannose, complex glycans (per-

Me)

Development of spin-column solid phase permethylation

(Kang, et al., 2008c)

Amyloidogenic 1 LC proteins Human

PNGase F, TOF, complex glycans (per-

Me), ESI-MS/MS

To investigate mechanisms leading to amyloidosis

(Connors, et al., 2007)

β-N-Acetyl-hexosaminidase

Aspergillusoryzae

PNGase F, Endo H, MALDI

Structural determination of high-mannose glycans and

biochemical studies

(Ettrich, et al., 2007)

Carbonic anhydrase IX

Human in sf9 cells or murine NS0 myeloma

cells

Trypsin, L-, R-TOF (DHB, THAP/NH cit),

glycopeptides

Structural determination. High mannose, N-linked only in sf9

cells. Mammalian cells produced O-linked glycan

(Hilvo, et al., 2008)

Carcino-embryonic

antigen

Human (liver metastasis of

colon carcinoma) PNGase F,TOF (DHB)

Structural determination and development of glycoblotting method for sample extraction.

(Furukawa, et al., 2008)

Cauxin Cat urine PNGase F, R-TOF (DHB), QIT-TOF,

glycans

Structural determination of bisected and complex glycans

(Suzuki, et al., 2007)

CD9P-1 Human, HEK-293 cells

PNGase F, R-TOF, glycans (Per-Me), exoglycosidases

Structural determination, > 40 high-mannose and complex

glycans

(André, et al., 2007a)

CD52 Human

PNGase F, TOF, TOF/TOF, (CHCA, DHB) ESI, glycans

(per-Me)

Structural determination. Complex glycans with N-Ac-

lactosamine repeats

(Parry, et al., 2007b)

CD-133 Human cord blood

PNGase F, R- and L-TOF, high-Man, hybrid,

complex glycans

To study differences in glycosylation in CD-133+ and

CD-133- cells

(Hemmoranta, et al., 2007)

CEACAM 1 Expressed in 293 cells

PNGase F, R-TOF (DHB), glycans, ESI-MS/MS (negative ion)

Structural determination as model for HIV. High-mannose

and complex glycans

(Scanlan, et al., 2007)

Coagulationfactor VII Human plasma

PNGase F, Endo H (on target), TOF (DHB, THAP, CHCA), LC-ESI-MS/MS, glycans

(free + 2-AB)

Structural determination of complex glycans (bi- and tri-

antennary)

(Fenaille, et al., 2008)

Complement factor H Human

PNGase F, Endo H, R-TOF (CHCA, DHB, THAP), LC-MS/MS

Peptides, glycopeptide

Site-specific structural determination of complex glycans. Glycan mass by

difference.

(Fenaille, et al., 2007b)

Egg-secreted protein

Schistosoma mansoni (eggs and cercarial

secretion)

PNGase F, PNGase A, TOF, TOF/TOF

(DHB), ESI-MS/MS, glycans (per-Me)

Structural determination of high-mannose and

paucimannosidic glycans

(Jang-Lee, et al., 2007)

4




Factor X activator

Daboiasiamensis

(Russel’s viper)

Trypsin, chymotrypsin, PNGase F, TOF,

TOF/TOF, Q-TOF (DHB), glycans (per-

Me)

Structural determination of 41 high-mannose and complex

(bi-, tri-, tetra-, penta-antennary) glycans

(Chen, et al., 2008a)

α-Fetoprotein Human fetal-cord serum

Trypsin, oxidation, TFA, TOF, TOF/TOF,

complex glycans

To evaluate chemical bonding for enrichment of

glycopeptides “reverse glycoblotting”

(Kurogochi, et al., 2007)

Fibrinogen Bovine pancreatic

Trypsin, oxidation, TFA, oxidation, TOF, TOF/TOF,

Chemical bonding for enrichment of glycopeptides

“reverse glycoblotting”


GP-140 HIV Trypsin, TOF (DHB/CHCA)

Optimization of methods for glycopeptide analysis

(Zhang, et al., 2008d)

Hemocyanin Rapana venosa TOF/TOF (DHB), PNGase F, glycans

(Amide derivs)

Structural determination. Complex and hybrid glycans. Chains with HexA-Fuc link

(Sandra, et al., 2007)

HIV Envelope proteins -

Trypsin PNGase F, TOF/TOF (DHB/

CHCA), LC-MS-ICR, Glycopeptides

Structural identification. High-mannose and complex (bi- and tri-antennary) glycans.

Inferred with “GlycoPep DB”

(Go, et al., 2008)

HIF envelope glycoprotein

CON-Sgp140∆ CFI

Recombinant

Trypsin, PNGase F, TOF/TOF

(DHB/CHCA), HPLC-ESI-FTICR

Comparison of techniques for site analysis, high-mannose, hybrid and complex (bi-, tri-

antennary)

(Irungu, et al., 2008)

ICAM-3 Human leukocytes

PNGase F, Trypsin, TOF (arabinosazone),

glycans

To study binding of DC-SIGN (through Lewis-X residues)

(Bogoevska, et al., 2007)

IgG Human serum

PNGase F (on-target), L-, R-TOF (ATT), complex glycans

(phenylhydrazones)

Development of method for on-target glycan release.

(Lattová, et al., 2007)

IgG Human B-cell receptor

PNGase F (in-gel), R-TOF (DHB), high-Man

glycans, HPLC

Identification of high-mannose glycans in antigen-binding site

in follicular lymphoma cells

(Radcliffe, et al., 2007)

IgG Mice PNGase, TOF, complex glycans (Per-Me)

Agalactosylated IgG antibodies depend on cellular

Fc receptors for in vivoactivity

(Nimmerjahn, et al., 2007)

IgG Human Trypsin, TOF (DHB/CHCA)



IgG Human PNGase F, TOF (DHB), complex

glycans (Girard’s T)

Use of Girard’s T reagent for quantification

(Gil, et al., 2008)

IgG1 Human in mouse myeloma

PNGase F, TOF, complex glycans (2-

AA)

High sialylation impairs functionality

(Scallon, et al., 2007b)

IgG1 Human PNGase F, TOF (s-DHB)

Removal of fucose produces enhancement of antibody-

dependent cellular cytotoxicity

(Matsumiya, et al., 2007)

IgG1 Mouse

Trypsin PNGase F, TOF, complex

(biantennary) glycans (Me ester, aoWR

derivative)

Impact of a three amino acid deletion in the CH2 domain of murine IgG1 on Fc-associated

effector functions

(Baudino, et al., 2008a)

glycans, glycopeptides

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IgG2a, IgG2b Mouse

Trypsin PNGase F, TOF, complex

(biantennary) glycans (Me ester, aoWR deriv)

Aspartic acid at Asn 265 in the CH2 domain crucial for Fc-associated effector functions

(Baudino, et al., 2008b)

IgG1, 2, 3, 4 Human PNGase F, TOF

(sDHB), complex glycans

IgG2 found to protect mice against Cryptococcus neoformans infection

(Beenhouwer,et al., 2007)

Insulin receptor Human PNGase F, TOF,

glycans (PMP derivatives)

Structures inferred from mass. To determine crystal structure

High-mannose, hybrid, complex glycans

(Sparrow, et al., 2008)

Interferon-γ Human in CHO cells and mutant

cells

Trypsin, PNGase F, TOF (DHB), complex

(bi-, tri-, tetra-antennary) glycans

(per-Me)

Mutant cell line, MAR-11 produces glycans without

Neu5Ac.

(Lim, et al., 2008)

Hemagglutinin

Avian H5N1 influenza virus recombinant,

HEK293F cells

Trypsin, PNGase F, TOF, TOF/TOF

(DHB), high-mannose, complex (bi-, tri-, tetra-ant) glycans (per-Me)

Proteins, as individual or oligomeric trimers can elicit potent neutralizing antibody

responses to avian H5N1 influenza viruses.

(Wei, et al., 2008a)

Histamine H1receptor

Human, recombinant, sf9

cells

Trypsin, TOF/TOF (DHB), LC-MS/MS,

glycopeptides

Structural determination of paucimannosidic and

functional analysis after baculovirus-driven and in vitro

cell free expression

(Sansuk, et al., 2008)

HMW1 Adhesin Haemophilus influenzae

Trypsin, Glu-C, Lys-C, TOF/TOF,

glycopeptides

Identification of Hex and Hex2attached to Asn in N-linked

consensus sequence

(Gross, et al., 2008)

Kringle 3 protein Pichia pastoris PNGase F, TOF, high-

mannose glycans

Identification of a new family of genes involved in β-1,2-

mannosylation

(Mille, et al., 2008)

Mannoproteins Saccharomyces cerevisiae

PNGase F, TOF (DHB), high-Man (Man8GlcNAc2)glycans (2-AP)

Deletion of genes encoding hypermannosylation impedes

growth

(Zhou, et al., 2007b)

Metallo-proteinase inhibitors

Galleriamellonella

PNGase F, TOF, glycans

Identification of two inhibitors encoded by metalloproteinase

gene. Man3GlcNAc2,Man3 2GlcNAc Fuc

(Wedde, et al., 2007)

Mucin Rat sublingual

PNGase F, TOF, TOF/TOF (DHB), glycans (per-Me),

GC/MS

Structural determination of hybrid glycans

(Yu, et al., 2008a)

Myelin protein zero (P0) Xenopus laevis

PNGase F (in-gel), TOF (DHB,

arabinosazone), ESI, MS/MS, glycans (per-

Me)

Structural determination of high-mannose and hybrid

glycans. Equilibrium between non-covalent dimer and

monomer

(Xie, et al., 2007)

NCAM Mouse brain

(WT plus mutants)

Trypsin, PNGase F, R-TOF/TOF (ATT,

ATT/NH4-Cit (-ve), DHB, CHCA), ESI,

glycans (2-AA, 2-AP) Glycopeptides

To evaluate contribution of the polysialyltransferases

ST8SiaII and ST8SiaIV to polysialic acid synthesis in

vivo

(Galuska, et al., 2008)




Nephrin Human PNGase F, R-TOF

(Glycopeptides, trypsin)

Site analysis. 9 of 10 sites occupied with complex and

high-mannose glycans

(Khoshnoodi, et al., 2007)

Nipah virus attachment

glycoprotein HEK293T cells

PNGase F, TOF/TOF (DHB), ESI-MS/MS

(-ve) glycans

Crystal structure and structure determination of high-

mannose, hybrid and complex (biantennary) glycans

(Bowden, et al., 2008)

NKp30 Recombinant PNGase F, (in-gel),

TOF (DHB), complex glycans, HPLC

Altered glycosylation of recombinant NKp30 hampers

binding to heparan sulfate

(Hershkovitz, et al., 2008)

Ole e 1 (Olive pollen antigen) Olea europaea

Trypsin, PNGase F, L-TOF, TOF/TOF

(CHCA), MS/MS, glycopeptides

Structural determination of hybrid, complex (assigned

from mass)

(Napoli, et al., 2008)

Ovalbumin Chicken

PNGase F (on-target), L-, R-TOF (ATT), complex glycans

(Phenylhydrazones)

Development of method for on-target glycan release.


Ovalbumin Chicken

Hydrazine, R-TOF (DHB), complex and high mannose glycans

(2-AP), HPLC

Development of gas-phase method for 2-AP

derivatization

(Nakakita, et al., 2007b)

Ovalbumin Chicken

PNGase F, TOF (DHB), high-mannose,

hybrid and complex glycans (Girard’s T

reagent)

Use of Girard’s T reagent for quantification

(Gil, et al., 2008)

P0 Bovine brain PNGase F, R-TOF

(DHB, CHCA), hybrid and complex glycans

Distribution in lipid rafts depends on glycosylation

(Fasano, et al., 2008)

Peroxidase Zinnia elegans FT-MS, R-TOF

(CHCA), peptides after deglycosylation

Structural determination of low-mannose and

paucimannosidic glycans. Deduced structures (see text)

(Gabaldon, et al., 2007)

Peroxidase Hordeum vulgare (Barley)

Trypsin, R-TOF, Glycopeptides

Glycoprotein expression in mature seeds

(Laugesen, et al., 2007)

Phytase Medicagotruncatula

PNGase A, TOF/TOF (DHB)

High-mannose and paucimannosidic glycans. To track subcellular localization

(Abranches, et al., 2008)

Pregnancy-associated

glycoproteins Pregnant cow

PNGase F, TOF/TOF (THAP/NH4-Cit -ve, DHB,+ve), glycans

(per-Me)

High-mannose, complex (bi-, tri-, tetra-antennary).

Identification of unusual tetra-antennary glycan with

bisecting GlcNAc

(Klisch, et al., 2008)

Protein C inhibitor Human plasma

PNGase F, R-TOF (DHB), TOF/TOF

(DHB, CHCA), glycans (per-Me)

Structural determination of complex glycans. All 3 sites (Asn-230, Asn-243, and Asn-

319 occupied)

(Sun, et al., 2008b)

Rhamnose-binding

glycoprotein

Spanish mackerel

(Scomberomorous niphonius)

PNGase F, TOF (DHB), glycans

Structural determination of complex and biantennary

glycans

(Terada, et al., 2007)

Ribonuclease B Bovine PNGase F, TOF/TOF (DHB), high-mannose

glycans



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An improvement on the simple Asn–Asp conversion is toperform the glycan release in the presence of a 1:1mixture of 16Oand 18O labeled water. The deglycosylation site can then readilybe identified by the doublet peaks separated by two massunits. This method was used by Sandra et al. (2007) to mapglycosylation sites in Rapana venosa hemocyanine. However,Angel et al. (2007), while using this procedure in a study onglycosylation of polygalacturonase-inhibiting protein isolatedfrom Pyrus communis (pear), noticed that 60% of asparaginesthat were labeled were not part of a consensus sequence and thus,should not have been glycosylated. The problem was traced to

residual trypsin that was used in the proteolysis still being activeat the deglycosylation stage and that this enzymewas responsiblefor incorporation of 18O into the C-terminus of the peptide.

As discussed earlier in this review, many investigatorsperform pre-digestion extraction of glycoproteins from protein/glycoprotein mixtures before further analysis. Calvano, Zambo-nin, and Jensen (2008) have compared lectin-affinity andhydrophilic interaction chromatography (HILIC), alone or incombination, for identification of glycosylation sites in serumglycoproteins. Eighty-six N-glycosylation sites in 45 proteinswere identified using a mixture of three immobilized lectins for


Ribonuclease B Bovine PNGase F, TOF/TOF

(deglycosylated peptides)

Shows that PNGase F can act up- or down-stream of protesomal degradation

(Kario, et al., 2008)

S19AHEK293T and GnTI-deficient HEK293S cells

Endo H, TOF (DHB), ESI, MS/MS

Reduction in glycosylation to aid crystallization

(Chang, et al., 2007a)

Secretedglycoprotein Zaire ebolavirus

Trypsin, TOF, Glycopeptides, ESI-

MS/MS

Structural and biophysical characterization. Site analysis

(Barrientos, et al., 2007)

S-Layerglycoprotein

Haloferax volcanii

Trypsin, TOF/TOF (CHCA), glycopeptides

Structural determination. Ident of 2nd glycosyltransferase

(Abu-Qarn, et al., 2008)

S-Layerglycoprotein

Haloferaxvolcanii

Trypsin, TOF/TOF (CHCA),

Glycopeptides

Study of the effects of deleting two genes on glycan assembly.

Pentasaccharide with Hex2,HexA2 +190 Da

(Abu-Qarn, et al., 2007)

sMIg HEK293 cells PNGase F,TOF (DHB), ESI, MS/MS

Disruption of α-mannosidase II induces formation of Man6GlcNAc2 hybrids

(Crispin, et al., 2007)

Surfaceglycoprotein Ebola virus

PNGase F, TOF/TOF (DHB), glycans (per-

Me

Structural determination of high-mannose and complex glycans. Structure deduced

from mass. LSECtin proposed as target

(Powlesland, et al., 2008)

Tamm-Horsfall protein Human urine

PNGase F, TOF (DHB), high-mannose and complex glycans

(Per-Me), HPLC

Effect of interstitial cystitis on N-glycans

(Parsons, et al., 2007)

T-Cell receptor HeLa cells

PNGase F, TOF/TOF (DHB), high-mannose and complex glycans

(Per-Me)

Effect of valosin-containing protein; p97 in the control of

N-glycosylation

(Lass, et al., 2007)

TIMP-1 Human Trypsin, Q-TOF (DHB, CHCA), glycopeptides

Development of gel-based method for glycoprofiling

(Thaysen-Andersen, et

al., 2007)

Transferrin Human Trypsin, TOF (DHB/CHCA)



Various enzymes

Chrysosporiumlucknowense

Trypsin, TOF (DHB, CHCA), Glycopeptides

Structural determination. Composition only

(Gusakov, et al., 2008)

YJL171c and GP38

Saccharomyces cerevisiae

PNGase F, TOF, glycans (per-Me)

Yeast engineered to produce only Man8GlcNAc2. Elicits

specific GP120-binding antibodies

(Luallen, et al., 2008)

1Format (not all items present): Glycan release method and/or protease, MALDI method (matrix), compounds run (derivative), other

methods.



consecutive glycoprotein enrichment. The combination of lectinaffinity enrichment of glycoproteins and subsequent HILICenrichment of tryptic glycopeptides identified 81 N-glycosyla-tion sites in 44 proteins following deglycosylation and observa-tion of the Asn–Asp conversion. Bunkenborg, Hagglund,and Jensen (2007) have also purified glycoproteins by lectinchromatography, digested them with trypsin and enriched theglycopeptides by HILIC chromatography. Rather than use of theAsn–Asp conversion, the glycan heterogeniety was reduced bytreatment with exoglycosidases and the resulting glycopeptideswere examined by MALDI-TOF and ESI mass spectrometry toreveal the glycosylation sites.

Two methods for identification of sialylated glycosylationsites have been reported by Ghesquiere et al. (2007). The first,called ‘‘direct sorting’’ involved a preliminary chromatographicstep followed by treatment with sialidase. The fractions from thisreaction were then re-chromatographed under identical con-ditions in a procedure known as diagonal chromatography.Fractionswere examined byMALDImass spectrometry to detectthe glycopeptides that had changed as the result of thedesialylation. In the second approach, called ‘‘indirect sorting,’’depleted serum was split into two equal parts. One part wastreated with sialidase and both samples were then digested withtrypsin. Peptides derived from the non-desialylated fraction werelabeled with 18O at the carboxy terminus to produce a massincrease of 4Da. Next, equal amounts of both digests weremixedand separated by RP-HPLC. At this stage, four types of peptidescould be distinguished with respect to N-glycosylation:18O-labeled sialylated peptides and their 16O-labeled desialy-lated counterparts, which would both elute from the column atdifferent times and could be collected in different fractions. Thethird class was composed of peptides that were glycosylatedbut not sialylated and these would not segregate into 16O- and18O-peptides. Finally, all remaining nonglycosylated peptidesshowed co-elution of their 16O- and 18O variants. Followingfraction collection, the peptides were N-deglycosylated usingPNGase F, a treatment that will not affect the HPLC retention ofnonglycosylated peptides in the second run. However, it willproduce chromatographic shifts for the first three peptide classesmentioned above, because glycans had been removed. 16O/18Ovariants of non-sialylatedN-glycopeptides co-eluted and showedsimilar ion intensities as isotopic couples, but in vivo sialylatedpeptides showed up as a single isotopic variant at differentelution times. Thus, the ‘‘indirect’’ sorting process identifiedsialylated peptides in retrospect based on three criteria: thechromatographic shift between sialylated and desialylatedpeptides, their appearance as isotopic singles (16O or 18O),and the Asn to Asp conversion in the consensus motif forN-glycosylation as the result of deglycosylation. Themethodwasused with immuno-depleted mouse serum to identified 93sialylated glycosylation sites in 53 serum proteins a result thatclaimed to be one of the largest hitherto reported catalogues ofin vivo sialylation sites of mouse serum proteins.

e. Glycan identification from glycopeptides. Because mostglycoproteins are too large for their attached glycans to beidentified, either they are cleaved into peptide fragments,commonly with trypsin or the glycans are removed for separateanalysis. If the mass of the glycopeptide is known, thecompositions of the attached glycans can be readily calculated.MS/MS and/or exoglycosidase digestions can then be used to

determine the detailed structure. An in-gel tryptic digestion hasbeen used by Thaysen-Andersen et al. (2007) followed bynanoscale HILIC chromatography to enrich the glycopeptides.Site-specific identification of N-glycans from TIMP-1 was usedto validate the method.

Pronase digestion. Several alternatives to trypsin cleavagehave been reported. One of these is to use the non-specificprotease, pronase to reduce the protein to a small peptide withup to six amino acids attached to the glycan (Yu et al., 2007d).DHB doped with ammonium tri-citrate increased the yield of[MþH]þ ions at the expense of [MþNa]þ. The [MþH]þ ionswere mainly associated with the peptide whereas the [MþNa]þ

ions were formed from the sugar. In an improved techniquethat avoids enzyme autolysis and offers a simplified clean-upprocedure, Clowers et al. (2007) have used immobilized pronaseand monitored the cleavage reaction over time. Although themethod was combined with ESI monitoring it should becompatible with MALDI-TOF analysis. One of the problemswith the pronase approach is the long reaction times (1–3 days)that are required. Liu et al. (2008b) have reduced this to as littleas 5min using microwave irradiation. Because of poorsignals associated with the presence of basic amino acids,the investigators methylated them with methyl iodide to givea quaternized amine group, thus considerably enhancing thesignal.

Acid cleavage. Acids such as acetic acid with microwaveassistance have been found to produce rapid cleavage of proteinchains at aspartic acid residues. In an extension to the originalreport, Li et al. (2008d) have examined the effect of the reactionon the integrity of glycans from glycoproteins.With ribonucleaseB, the glycosidic bond to asparagine was found not to be cleavedunder the hydrolysis conditions whereas theO-linked chain froma-crystallin A was cleaved. Hydrolysis within the carbohydratechains was minimal with the N-glycans.

Isolation of glycopeptides. As discussed above, glycopep-tide analysis in the presence of peptides is difficult because of thegenerally weak signals given by the glycopeptides. To addressthe problem, Kanie et al. (2008) have developed an HPLCfractionation technique to prepare samples for MALDI-TOFanalysis. The first step involved rough fractionation using areversed-phase (RP) cartridge column under acidic conditions todecreases the number of peptides in a tryptic digest. In a secondstep, comparative RP-HPLCwas performed using phosphate andborate buffers under basic conditions and the two chromatogramswere compared to differentiate glycopeptides from peptides.Zhang, Go, and Desaire (2008d) have optimized factors such assample preparation, pre-mass spectral separations, mass spectraltechniques and data analysis. Best results were obtained byHPLC coupled with pre-fractionation of peptides and glycopep-tides. It was emphasized that mass spectrometry should beperformed in both positive and negative ion modes for bestcoverage. By using optimized techniques, over 300 glycopep-tides were identified from the HIV envelope glycoprotein GP-140, a molecule containing 27 potential glycosylation sites.Fukuyama et al. (2008) have found that their newly synthesizedliquid matrix, G3CA (see above) not only preserves the integrityof sulfated and sialylated carbohydrates but also, with ribonu-clease B digests at least, produces preferential ionization ofglycopeptides, particularly in negative ion mode. Picariello et al.(2008) have made a study of N-linked glycoproteins in humanmilk. Glycopeptides were selectively enriched from tryptic

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digests by hydrophilic interaction LC and glycan-free peptideswere obtained by PNGase F digestion. Samples were examinedby MALDI-TOF/TOF MS and, assisted by the Asn–Aspconversion, 32 different glycoproteins containing 63N-glycosylation siteswere identified. Theglycoproteins includedimmunocompetent factors, membrane fat globule-associatedproteins, enzymes involved in lipid degradation and cell differ-entiation, specific receptors, and other gene products with stillunknown functions.

Kubota et al. (2008) have used a combination of celluloseand lectin chromatography to fractionate glycopeptides prior toMALDI-TOF andMALDI-QIT-TOF analysis, both to extract theglycopeptides and to obtain structural information. A mixture ofhuman serum transferrin and bovine RNase B was digested withLys-C. Glycopeptides were extracted from the resulting peptide/glycopeptide mixture; sialylated biantennary glycopeptidesfrom transferrin were obtained by Sambucus nigra agglutinin(SNA) chromatography whereas the high-mannose glycansfrom RNase B were recovered by concanavalin A (Con A)chromatography. Spectra of the sialylated glycans recorded withthe MALDI-TOF instrument contained the molecular ion fromthe expected disialylated biantennary glycan-containing glyco-peptide but also some desialylated product and a series of cross-ring cleavage products. The corresponding MALDI-QIT-TOFspectrum contained none of the disialylated molecular ions,an observation consistent with earlier reports of considerablefragmentation produced with this instrument. Fortunately, laterversions of the instrument are not subject to this fragmentationproblem.

Tzur, Markovich, and Lichtenstein (2008) have developed a2-D array for sequencing both N- and O-glycans. The firstdimension of the array consisted of several lectins that wereimmobilized onto theMALDI plate via a biotin-streptavidin link.The second dimension consisted of a series of exoglycosidasesand trypsin. Trypsin and exoglycosidase digestions wereperformed on the glycoproteins and the products were appliedto the array. Glycans adhered to the lectins and the unglycosy-lated peptides were washed away. Finally, the matrix, DHB, wasadded to the sample spots and the glycopeptides were examinedby MALDI-TOF MS. The well-characterized glycoprotein,ribonuclease B (RNAse B), was used to test the system andapo-transferrin was used to illustrate the identification ofglycosylation sites. To confirm the results, the glycans wereremoved with PNGase F, labeled with 2-AB and profiled byHPLC. Finally, the array was used to analyze glycosylationof a recombinant fusion natural killer receptor (NKp46D2-Ig)produced in CHO cells.

Glycan composition by mass difference. As an alternativeto working with the intact glycopeptide, the mass of a glycan canbe determined by comparing the masses of a glycopeptide beforeand after deglycosylation. As an example of the latter technique,glycan masses and constituent monosaccharide compositionsfrom peroxidise extracted from the plant Zinnia elegans havebeen deduced from measurements of tryptic glycopeptidesbefore and after deglycosylation with trifluoromethanesulfonicacid (TFMS) (Gabaldon et al., 2007). By the application of rulessuch as the conserved nature of the constituent monosaccharidesand the known presence of the trimannosylchitobiose core,together with information on known structures from plants, theauthors were able to propose the presence of low-mannose(Man3-4GlcNAc2) and paucimannosidic structures.

A comparative study of HPLC/ESI-FTICR MS versusMALDI-TOF/TOF MS for the analysis of the highly glycosyl-ated HIV envelope glycoprotein protein, CON-S gp140DCFIhas been made. The results showed that ESI-MS/MS providedthe most complete information on the glycan moieties present,including glycans containing acidic monosaccharide units,whereas MS/MS with the MALDI-TOF/TOF spectrometer wasbetter at providing information on the peptide portion of thesame glycopeptide. Information from the two techniques wascomplementary. Only 15 of the potentially glycosylated siteswere identified by both techniques but a further 3 and 6 siteswere uniquely identified by ESI and MALDI-TOF/TOFrespectively, allowing 24 of the 31 sites to be identified.However, to achieve 100% glycosylation coverage, deglycosy-lation experiments and lower resolution MALDI MS wererequired.

f. Glycan release. N-glycans can be released either chemicallyor enzymatically but, whereas chemical methods were oncedominant,most investigators nowuse enzymes as reflected by theentries in Tables 4 and 5.

Chemical release. The most common chemical releasemethod is still hydrazinolysis with anhydrous hydrazine (H2N-NH2). A reassessment of the hydrazinolysis method for glycanrelease has been made by Anumula (2008). Optimum releaseconditions were 608C for O-linked glycans and 958C for acombination of O- and N-linked glycans with a reaction time of6 hr. Some enhancement ofN-glycan release at low temperatureswas found if the hydrazine was diluted with methylhydrazine(MeNH-NH2). It was recommended that polypropylenereaction tubes with O-ring seals be used because PTFE-cappedglass vials were found to produce considerable sialic acid loss(from fetuin). Following evaporation of the hydrazine, thereleased glycans were derivatized with 2-AA concurrently withthe re-acetylation stage. Practical details for hydrazinolysishave been published in a book on Experimental Glycosciences(Nakakita, 2008).

As an alternative to the use of anhydrous hydrazine,Nakakita et al. (2007a) have developed amethod using hydrazinehydrate which they claim is safer and which achieves 80% yieldscompared with traditional hydrazine release. The glycoproteinand hydrazine hydrate were heated at 908C for 10 h. afterwhich time the hydrazine monohydrate was removed in vacuousing a rotary pump connected to a cold trap. The liberatedN-glycan were re-N-acetylated by a standard procedure. Themethod was applied to egg yolk proteins with the productsanalyzed by MALDI-TOF MS as their 2-AP derivatives. Sialicacids in a1! 6-linkage were stable under these conditions.

Enzymatic release. The popular enzyme, PNGase F, hasbeen shown to cleave glycans as short at chitobiose but only inthe presence of a hydrophobic region in the protein near to theglycosylation site (Hagihara et al., 2007). The study also showedmore rapid proteosomal degradation of proteins after in vivoremoval of the glycans with PNGase. Takahashi, Yagi, and Kato(2008b) have compared PNGase A and F for their ability torelease N-glycans and have published experimental details of amethod using PNGase A. PNGase F will not release glycanscontaining a fucose residue attached to the three-positionof the reducing-terminal GlcNAc residue because of a stericinteraction. Neither will it release glycans from many nativeglycoproteins, such as ribonuclease B unless these are first



TABLE 5. Use of MALDI MS for examination of N-glycans from intact organisms, tissues, or protein mixtures

Organism Type Methods1 Notes Reference

Achatina fulica (African giant

snail)Eggs

PNGase F, TOF/TOF (DHB), ESI-MS, glycans

(2-AP)

Structural determination of high-mannose, complex (di-, tri-,

tetra-antennary, truncated) glycans. No fucose

(Park, et al., 2008c)

Arabidopsisthaliana Flowers

PNGase F, TOF/TOF (DHB),

high-mannose, paucimannosidic

glycans

Extension of in-gel release method for small tissue samples

(Rendic, et al., 2007a)

Arabidopsisthaliana

Total glycoproteins

Hydrazine, TOF, glycans (2-AP), Exoglycosidase

Structural determination of paucimannosidic glycans from suspension-culture in MM2d

cells

(Fujiyama, et al., 2007a)

Arabidopsisthaliana Leaves

PNGase A, TOF (DHB), high-mannose and

paucimannosidic glycans

Identification of the gene encoding the α1,3-

mannosyltransferase (ALG3)

(Henquet, et al., 2008)

Ascaris suum Total extract PNGase A, PNGase F, TOF

MALDI used to determine products of fucosyltransferase

(Poltl, et al., 2007)

Bos primigenius(cow)

Lung (acetone powder)

Hydrazine, TOF (DHB), glycans (2-

AP)

Structural determination of sulfated N-glycans

(Murakami, et al., 2007)

Biomphalaria glabrata

(Freshwater snail)

Hemolymph

Trypsin, PNGase F, R-TOF (ATT), glycans (2-

AP

Characterization of 116 complex and paucimannosidic glycans

that cross-react with Schistosoma mansoni

glycoconjugates. Some contained 3-O-Me-Man

(Lehr, et al., 2007)

Caenorhabditiselegan Total extract

PNGase A, TOF (ATT),

paucimannosidic glycans (2-AP)

Investigation of the role of N-Ac-hexosaminidases in

biosynthesis

(Gutternigg, et al., 2007b)

Caenorhabditiselegan Total extract

PNGase F, TOF/TOF (DHB), high-mannose and paucimannosidic

glycans



Caenorhabditiselegans

Glycansrecognized by

C. elegans galectin LEC-6

Hydrazine, TOF/TOF (DHB), glycans (2-AP), Exoglycosidase

Structural determination. Man3GlcNAc2 with fucose and Fuc-Gal in various antenna and

core positions

(Takeuchi, et al., 2008)

Caenorhabditiselegans mutant Total extract

PNGase F, PNGase A, R-TOF

(DHB), glycans (per-Me)

Bre-1 Mutant produces glycans deficient in fucose. Composition

only

(Barrows, et al., 2007)

Chicken and quail

Colon epithelial cells

PNGase A, L-TOF (DHB), glycans (2-

AP)

Glycans (complex di, tri-antennary, sialylated and bisect) have sialyl groups responsible

for binding of influenza A viruses to human-type receptors

(Guo, et al., 2007)

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Ciona intestinalis(sea squirt)

Larvae (various tissues)

PNGase A, TOF, TOF/TOF (DHB), glycans (2-AP), LC-ESI-MS/MS (glycoproteins)

Structural determination of paucimannosidic and high-

mannose glycans. First ident. of paucimannosidic glycans with

xylose in this species

(Yagi, et al., 2008a)

Collocalia (swift) Saliva (Edible bird’s nest)

Trypsin, glycoamidase A,

TOF/TOF (DHB), GC/MS, glycans

(2-AP)

First analysis of N-glycans.Suggested that glycans inhibit

influenza virus infection. Complex, α2→3-linked

sialylated bi-,tri-, tetra-antennary

(Yagi, et al., 2008b)

Drosophilamelanogaster Embryo

PNGase A, PNGase F,

(Trypsin), L-, R-TOF (DHB),

glycans (2-AP, per-Me),

Exoglycosidase

Structural determination of high-mannose and complex glycans

(Aoki, et al., 2007)

Drosophilamelanogaster

Heads, bodies, cell lines


high-mannose glycans



Entamoebahistolytica Whole cells

Trypsin PNGase F, TOF (DHB),

glycans (per-Me), ESI, MS/MS

Identification of unique glycans containing galactose and glucose

(Magnelli, et al., 2008)

Gastropods(various) Skin, viscera

PNGase A, pepsin, thermolysine, TOF (DHB, ATT (on-

target exoglycosidase

digestion), glycans (2-AP)

Structural determination of high-mannose and paucimannosidic

glycans, mainly with terminal 3-OMe-mannose Very complex

glycosylation

(Gutternigg, et al., 2007a)

Human Dendritic cells PNGase F, TOF,

TOF/TOF (DHB), glycans (Per-Me)

Study of glycosylation changes during cell maturation

(Bax, et al., 2007)

Human HeLaS3 cells Hydrazine, TOF

(DHB), glycans (2-AP)

Method for detection of weak mannose binding

(Kawasaki, et al., 2007b)

Human Sperm PNGase F, TOF,

TOF/TOF (DHB), glycans (per-Me)

Glycans inhibit antigen-specific responses to sperm cells. High-mannose and complex glycans,

much fucose

(Pang, et al., 2007)

Human Stem cells PNGase F, TOF

Detection of Neu5Gc as contaminaton. Largely

reversible. complex glycans (composition only)

(Heiskanen, et al., 2007)

Human Bronchial epithelial cells

PNGase F, TOF, TOF/TOF (CHCA),

glycans (2-AB)

Avian H5N1 virus hemagglutinin found to require a2→6-sialic acid on long Gal-GlcNAc carbohydrate chains

(Chandrasekaran, et al., 2008)

Human Serum glycoproteins

PNGase F, (in gel), TOF

(DHB), HPLC, glycans (free and 2-

AB)

Development of in-gel release method, mainly HPLC analysis

(Royle, et al., 2008)





PNGase F, TOF/TOF (DHB)




PNGase F, R-TOF (DHB), ESI-

MS/MS (-ve ion)

Method for N-glycan structure determination, mainly -ve

MS/MS

(Harvey, et al., 2008a)

Mammillariagracillis (cactus)

Shoot, callus, tumor,

hyperhydric regenerant

PNGase A (in gel, 1-D), TOF (DHB),

glycans

Structural determination. Paucimannosidic (shoot, callus),

hybrid (tumor) and complex (regenerant) N-glycans vary with developmental stage of the plant

(Balen, et al., 2007)

Miniature pig Kidney


(per-Me and free), ESI (-ve ion)

α-Galactose identified on several complex glycans

(Kim, et al., 2008e)

Miniature pig Kidney


(Girard’s T reagent)

Use of Girard’s T reagent for quantification. (High-mannose, hybrid and complex glycans)

(Gil, et al., 2008)

Mouse lacking α1-2-

mannosidase 1B Various tissues PNGase F, TOF

(DHB) Inactive α1-4-mannosidase 1B

disrupts pulmonary development (Tremblay, et al.,

2007)

Mouse (diabetic) human Liver

TOF (DHB, PNGase F, CHCA),

glycans (2-AP, HPLC

Increase in core fucosylation of complex glycans in type II

diabetes

(Itoh, et al., 2007)

Mus musculus(Mouse) Brain

PNGase F, TOF (DHB), TOF-TOF

(DHB, CHCA), Glycans (per-Me)

Combination with method involving ceramide glycanase release for glycosphingolipids

(Parry, et al., 2007a)

Mus musculus(mouse) Brain


glycans



Mus musculus(mouse) T-Cells PNGase F, TOF,

glycans (per-Me) Effect of GlcNAc branching on

demyelination (Lee, et al.,

2007e)

Mus musculus(mouse)

mgat5+/+ and mgat5-/- tumor

cells

PNGase F, NaBH4, TOF, QIT-

TOF (DHB), glycans (per-Me)

Complex N-glycan number and branching shown to regulate cell proliferation and differentiation

(Lau, et al., 2007)

Nicotianabenthamiana Flowers


glycans



Oryctolagus cuniculus (rabbit,

New Zealand white)

Erythrocytes

PNGase F, TOF (DHB), TOF-TOF

(DHB, CHCA), glycans (per-Me)

Structural determination. To investigate why mouse sperm binds to rabbit erythrocytes. Complex glycans with high

degree of branching

(Sutton-Smith, et al., 2007)

Rat Liver plasma membranes

PNGase F, TOF (DHB), glycans (2-

AB), HPLC

No change in complex N-glycans with increased UDP-

sugar concentration

(Renner, et al., 2007)

Saccharomyces cerevisiaemutants

Whole organism

PNGase F, TOF (DHB) glycans

(2AP)

Disruption of MNN1 and OCH1 prevents hypermannosylation of

Man8GlcNAc2

(Zhou, et al., 2007c)

Saccharomyces cerevisiaemutants

Whole organism

PNGase F, TOF (DHB) glycans

(2AP)

Mutants prepared to investigate the response to temperature

(Zhou, et al., 2007b)

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denatured. However, ribonuclease BS that lacks amino acids 1–20 is sensitive due to the more flexible nature of the protein chain(Blanchard et al., 2008a).

Cleavage of the glycoprotein into peptides or denaturing itby use of detergent or by reduction and alkylation is an importantstep in glycan release to ensure that all of the glycosylation sitesare accessible to the enzyme. Kita et al. (2007b) have performedan extensive study on the release of N-glycans from humanserum glycoproteins in order to ensure reproducible andquantitative conditions for use in biomarker identification. Theefficiency of N-glycan release was found to differ considerablydepending on the conditions used (reducing agents, surfactants,protease treatment, or combinations of pre-treatments prior toPNGase F digestion). Optimum conditions were as follows:serum was mixed with 4 mL of 100mM ammonium bicarbonate(pH approx. 7.8), 16 mL of the surfactant 2-hydroxyl-3-sulfopropyldodecanoate (43, 0.5% in H2O) and 8 mL of 50mMdithiothreitol (44) followed by incubation at 558C for 45min toreduce the protein. Five mL of 135mM iodoacetamide (45) inH2O was added to alkylate the protein and the mixture wasallowed to stand at room temperature for 45min. After that time,5 mLof trypsin (400U)was added and themixturewas incubatedat for 1 hr at 378C followed by heat denaturation at 808C for15min. The serum samples were then incubated with PNGase F(2U) at 378C for 24 hr followed by heat denaturation at 908Cfor 15min. The final volume of all samples was adjusted to200 mL with 100mM ammonium bicarbonate. Sialic acidswere converted to methyl esters and analysis was by MALDI-TOF/TOF.

Celik et al. (2007) have used SDS to denature humanerythropoietin (EPO) produced in Pichia pastoris prior todeglycosylation by PNGase F and have noted that with 0.01%SDS, the major glycan Man17GlcNAc2 could be detected byMALDI-TOF, whereas when 0.1% SDS was used, only thesmaller Man9–11GlcNAc2 glycans were observed.

Planar SDS–PAGE, capillary-gel electrophoresis (CGE)on-a-chip, and MALDI-TOF mass spectrometry have beencompared for analysis of the enzymatic de-N-glycosylation ofantithrombin III and coagulation factor IX with PNGase F(Muller et al., 2007). Although all three methods worked well,the MALDI technique provided the most accurate molecular


Salmo salar (Atlantic salmon) Serum

PNGase F, TOF (DHB (1,4-isomer

stated), glycans (per-Me)

Mono-Ac sialic acids normal. Increased O-Ac under stress

(Liu, et al., 2008c)

Toxoplasma gondii

Glycoproteins from extract

TOF (DHB), PNGase A, glycans

(per-Me, 2-AB)

Parasite transfers glycans from host onto its proteins

(Garénaux, et al., 2008)

Toxoplasma gondii

Tryptic glycopeptides


(per-Me)

High-mannose N-glycans probably important in host cell

invasion

(Fauquenoy, et al., 2008)


methods.



weights; those determined by the other techniques were too highbecause of the attached glycans.

Various modifications aimed at speeding up release havebeen described. A method for PNGase F-release of N-glycansassisted by microwave irradiation has been reported to be muchfaster than conventional releasewith no detrimental effects on theglycan or residual protein. Glycans could be released in fewerthan 30min (Sandoval et al., 2007). A study of the use ofcombined aqueous/organic solvents has shown that PNGase Fremains active in solutions containing up to 60% acetonitrile.Under these conditions, deglycosylationwasmuchmore efficientthan when reactions were conducted in water or buffers. Mostglycoproteins could be deglycosylated in 1 hr at 378C and,furthermore, because of the absence of salts, etc., the mixturecould be examined directly by MALDI (Fenaille et al., 2007a).

Three publications (Furukawa et al., 2008; Miura &Nishimura, 2008b; Miura et al., 2008) have described a blottingmethod combined with chemical derivatization for extractingPNGase F-released glycans from serum for examination byMALDI-TOF MS. Furukawa et al. (2007b) have captured theglycans with a solid-phase hydrazide-functionalized glycoblot-ting polymer (BlotGlyco H) followed by washing to remove thenon-bound material. Unreacted hydrazide groups were blockedwith acetic anhydride and sialic acids were stabilized by methylester formation using 3-methyl-p-tolyltriazine (23). The blottedglycans were recovered in the form of their reducing-terminalderivatives by adding the aminooxy-containing compoundaoWR or the hydrazine-containing compound anthraniloylhydrazine (46). The procedure was said to take 8 hr in a 96-well plate format and was used with MALDI-TOF MS to detectover 120 N-glycans from human carcinoembryonic antigens. Inan alternative procedure described by Miura and Nishimura(2008b) and Miura et al. (2008), an anthranilic acid tag wasincorporated into the linker and the glycan and tag were releasedby cleavage of the linking disulfide bond with DTT. The methodwas applied to the detection of congenital disorders ofglycosylation, hepatocellular, and prostate cancer.

Experimental details for a more traditional solution releasehave also been published in connection with a study of humanaging (Vanhooren et al., 2008).

In-gel and related release methods. A method based on thetechnique introduced by Kuster et al. (1997) for in-gel release ofN-glycans with PNGase F but using a 96-well plate format hasbeen described (Royle et al., 2008). Glycoproteins were reducedand alkylated and set into SDS–PAGE gel blocks. PNGase F wasdiffused into the blocks, whichwere incubated overnight at 378C.Released glycans were extracted and either examined directlyby MALDI-TOF MS and negative ion ESI-MS/MS or labeled

with 2-AB and profiled by HPLC. Structural determinationrelied mainly on results from exoglycosidase digestion withthe resulting profiles analyzed with the aid of a database ofretention times (expressed as glucose units) called GlycoBase.This software can be found at http://glycobase.ucd.ie/cgi-bin/profile_upload.cgi. The method was used to characterize over100 N-glycans from human serum glycoproteins and to examinechanges in glycosylation in rheumatoid arthritis patients.It was claimed that 96 samples could be processed in 4 daysusing HPLC analysis but this could be shortened considerably ifMALDI-TOF was used for the analysis.

In another modification to the original in-gel release method(Kuster et al., 1997) for use with very small amounts of tissue,Rendic et al. (2007a) have ground the tissue directly in Laemmlisample buffer and briefly subjected the mixture to discontinuousTris-glycine-SDS–PAGE separation. Glycans were releasedwith PNGase F from the Coomassie-stained band that containedthe majority of proteins and characterized by MALDI-TOF. Themethod was successfully used to characterize N-glycans fromseveral nematode, plant insect and mammalian species (detailsare in Table 5). The authors commented that, although MALDI-TOFanalysisworkedwell, they doubted if therewould be enoughmaterial in the samples for fluorescent labeling and conventionalHPLC analysis.

Kimura et al. (2007) have developed an on-membranemicro-method for glycoprotein and N-glycan analysis. Reducedand alkylated proteins from albumin and IgG-depleted serumwere separated on a 2D SDS–PAGE gel and blotted onto anImmobilon PSQ PVDF membrane using established techniquesand stained using Direct Blue 71. The membranewas attached tothe MALDI target of an Axima QIT-TOF instrument and achemical ink-jet printer was then used, first to pre-wet themembrane with a solution of 0.25% (v/w) polyvinylpyrrolidone(PVP360) in 60% methanol or 0.25% (w/v) n-octyl-a-D-glucopyranoside (3/10) and then to deliver either trypsin ora mixture of PNGase F and neuraminidase to a small areaof each protein spot. After a 16 hr incubation (168C for trypsin,378C for the other enzymes), the inkjet printer was used tospray a few nanoL of DHB and positive ion MALDI spectrawere acquired with the QIT-TOF instrument. Proteins wereidentified by conventional peptide mass fingerprinting andN-glycans were identified from IgA1, transferrin and a1-macroglobulin.

On-target methods. Lattova et al. (2007) have developed amethod for glycan release and MALDI analysis of N-glycansfrom both glycoproteins and tryptic glycopeptides. Deglycosy-lation on the MALDI target and concomitant phenylhydrazoneformation was achieved in 1 hr with PNGase F and a mixtureof ATT matrix and phenylhydrazine (4/11) hydrochloride.The method was successfully applied to N-glycans from IgG,transferrin and chicken ovalbumin. ATT was also used byGutternigg et al. (2007a) as the matrix for performing on-targetexoglycosidase digestions of 2-AP-derivatized high-mannoseand paucimannosidic N-glycans from various gastropods.

g. N-Glycan profiling and structure determination of releasedglycans. The results of a comparative study by 20 laboratories onthe composition of two libraries of released glycans (transferrinand IgG) organized by the Human Proteome Organization(HUPO) Human Disease Glycomics/Proteome Initiative hasbeen published (Wada et al., 2007b). The best quantitative

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data were obtained by MALDI-TOF MS on permethylatedoligosaccharides (six laboratories) with results agreeing wellwith HPLC measurements made from reductively aminatedglycans. The results of the study endorse the power of massspectrometry-based methods for glycan identification andquantitation.

As mentioned above, N-glycans containing sialic acid areunstable under MALDI conditions but can be stabilized bymethyl ester formation or permethylation. Some investigatorsclaim that permethylation preceeding MALDI gives improvedperformance. Certainly, with reasonable amounts of sample, thiscan be the case, particularly for quantitation. However, sidereactions and problemswith sample clean-up can outweigh theseadvantages for very small sample amounts. Furthermore, if MS/MS in negative ionmode is contemplated, permethylation shouldbe avoided because the very specific fragmentation, discussedabove, relies on proton removal from underivatized hydroxylgroups.

Although MALDI-TOF was the preferred method forN-glycan profiling in a recent study by Harvey et al. (2008a),the best structural data were obtained by negative ion nanosprayCID. These spectra containedmany cross-ring fragment ions thatprovided specific information on structural features such asantenna structure, fucose location and the presence or absence ofbisecting GlcNAc that was not available from the correspondingpositive ion spectra. Although good correlation was foundbetween profiles recorded by ESI and MALDI, the ESI spectracontained additional ions produced by fragmentation andmultiple charging.

In some recent experiments on released glycans, stableisotope labeling has been used by Ito et al. (2007a) to determinethe sites of attachment of 1! 4-linked b-galactose to a tetra-antennary glycan (47). The glycan was synthesized enzymati-cally with 13C6-GlcNAc in each of the antennae. Thesecompounds were then allowed to react with b-galactose in thepresence of the galactosyltransferase and the products, as 2-APderivatives were separated by HPLC and examined by MALDI-MS/MS ([MþNa]þ ion). The sites of galactose attachment weredetermined by observingwhether or not theB-cleavage ions [M–GlcNAc]þ and [M–GlcNAc–Gal]þ did, or did not contain thelabeled GlcNAc. Another recent method uses partial acidhydrolysis (HCl, TFA, or H3PO4) assisted by short (30 sec)exposure to microwaves in a microwave oven to acquirestructural information fromN-glycans (Lee et al., 2008a). Bovinepancreatic RNase B, egg white avidin, human a1-acid glyco-protein and horseradish peroxidase were examined as examples.Capillary electrochromatography (CEC) of N-linked oligosac-charides from ribonuclease B in porous polyacrylamide mono-lithic columns has shown separation of the isomers ofMan7GlcNAc2. Glycans were monitored by MALDI (Gurycaet al., 2007).

h. Applications of MALDI to the detailed structural determi-nation of N-linked glycans. MALDIMShas been used in a largenumber of studies on the structure ofN-glycans during the reviewperiod. These are listed in Table 4 (from specific glycoproteins)and 5 (glycoprotein mixtures, tissues and whole organisms).Only a few of the more unusual studies and studies that identifynew glycans are mentioned here. N-glycan analysis is also ofimportance in the development of biopharmaceuticals; studiesin this area are listed in Table 6.

N-linked glycans from mammalian species. Sulfatedbiantennary glycans have been identified in glycoproteins frombovine lung (Murakami et al., 2007). The compounds werereleased by hydrazinolysis, and analyzed by partial acidhydrolysis, methanolysis, exoglycosidase digestion, variousforms of chromatography and MALDI-TOF analysis of 2-AP-labeled glycans. Identified compounds were based on thefucosylated biantennary core with the sulfate group locatedat the 6-position of the GlcNAc residue of the 6-antenna. Thedisialylated analogue was the dominant species.

N-glycans with very highly branched antennae terminatingin a-galactose have been identified on rabbit erythrocytes(Sutton-Smith et al., 2007). The compounds were examined byboth MALDI-TOF and MALDI-TOF/TOF MS from DHB orCHCAas their per-methyl derivatives and yieldedmasses of up tom/z 9,000. Highly fucosylated complex glycans have beencharacterized in human sperm as per-methylated productsby MALDI-TOF/TOF and combined gas chromatography/mass spectrometry (GC/MS). Tetra-antennary glycans with upto nine attached fucose residues (48) were identified (Panget al., 2007).

The presence of a biantennary, bisectedN-glycan containinga galactose residue attached to the bisecting GlcNAc (49), firstreported by Takegawa et al. (2005) from IgG, has also beenidentified byHarvey et al. (2008b) as part of a study into negativeion MS/MS for the differentiation of isomeric triantennary



TABLE 6. Use of MALDI analysis to monitor N-glycosylation in biopharmaceuticals

Biopharmaceutical Expression system Methods1 Notes Reference

Alkaline phosphatase CHO cells

PNGase F, TOF (DHB), high-Man

and complex glycans, HPLC

Effect of culture conditions. Glycan structure inferred by “GlycoMod”

(Nam, et al., 2008)

Chorionic gonadotropin

Human in Pichiapastoris

PNGase F, TOF (DHB, +ve, THAP,

-ve), glycans, (HPLC, 2-AB)

Structural characterization of high-mannose glycans (Man8-Man )15

(Blanchard, et al., 2007)

Chorionic gonadotropin Human urine

PNGase F, L, R-TOF (DHB), high-

Man, hybrid, complex (2-AB)

Comparison with glycans from recombinant hormone in other

species for identification in doping.

(Ramírez-Llanelis, et al., 2008)

Chorionic gonadotropin (15N-labelled)

Pichia pastoris

PNGase F. Endo H, trypsin, TOF

(CHCA, sinapinic), glycopeptides

Of two strains, GS115 and X-33, only glycosylation by GS115 found

suitable for production of biopharmaceuticals

(Blanchard, et al.,

2008b)

Erythropoietin Human in moss Physcomitrella∆ ∆ -fuc-t -xyl

PNGase A, (trypsin), TOF, glycopeptides,

glycans

EPO synthesis in a moss that avoids addition of antigenic plant-type

glycans

(Weise, et al., 2007)

Erythropoietin Human in CHO cells

PNGase F, TOF (DHB), complex glycans (2-AB)

Mn2+ addn. to culture improved galactosylation

(Crowell, et al., 2007)

Erythropoietin Human in goat mammary glands

PNGase F, R-TOF (DHB), -ve ion

ESI-MS/MS

Structural determination of complex glycans. Polyfucosylation found

(Sánchez, et al., 2007)

Erythropoietin Pichia pastoris PNGase F, TOF (s-DHB)

Structural determination of high-mannose glycans (Man17GlcNAc2)

(Celik, et al., 2007)

Erythropoietin CHO cells

Trypsin, oxidation, TFA, oxidation,

TFA, TOF, TOF/ TOF

To evaluate chemical bonding for enrichment of glycopeptides

“reverse glycoblotting”


Erythropoietin (rEPO-P, rEPO-

T, NESP)

Recombinant human

(commercial)

PNGase F (in-gel),TOF (DHB), complex glycans (2-AB), HPLC

To evaluate method based on HPLC, MALDI-TOF, exoglycosidase

digestion

(Llop, et al., 2007)

Erythropoietin Human in kidney

fibrosarcoma cells

PNGase F, TOF (DHB), complex glycans (2-AB)

Sodium butyrate (used to increase protein expression) promotes Gal-

GlcNAc extensions to glycan antennae. No effect on sialylation

(Crowell, et al., 2008)

Erythropoietin Human in goat milk

PNGase F, TOF (DHB) glycans (4-AB), ESI-MS/MS

(-ve), LC-MS

Structural determination. Monosialylated biantennary N-

glycans containing GalNAc–GlcNAc antennae predominate when human

EPO expressed in goat milk.

(Montesino, et al., 2008)

Erythropoietin (epoetin delta,

Dynepo)

Human fibrosarcoma cell

line HT-1080

PNGase F (in gel and solution), TOF (DHB (glycans),

sinapinic (proteins)), glycans

(2-AB), HPLC

Comparison with EPO produced in CHO cells. No Neu5Gc of Gal-GlcNAc extensions to antennae. complex (tri-, tetra-antennary)

glycans

(Llop, et al., 2008)

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Fas ligand Human in Pichiapastoris

Glycoprotein, L-TOF (sinapinic)

Truncation of N-terminus of protein and deletion of two N-sites to

improve yield

(Muraki, 2008)

Gluco-cerebrosidase Carrot cells

PNGase A, TOF, glycans (Per-Me),

FAB

To produce enzyme with mannose-terminating N-glycans

(Shaaltiel, et al., 2007)

Gluco-cerebrosidase Various PNGase F, TOF

No pharmacokinetic or pharmacological advantage of increased mannose residues

(Van Patten, et al., 2007)

α-Glucosidase Transgenic rabbit milk

PNGase F, TOF (DHB+,

THAP/NH Cit-), glycans

Structural determination of complex, hybrid and high-mannose glycans.

Glycoprotein for treatment of Pompe disease

(Jongen, et al., 2007)

House dust mite allergen, Der p1

Transgenic rice seed

Hydrazine, TOF, high-mannose glycans (2-AP)

To develop vaccine for house dust mite allergy

(Yang, et al., 2007)

IgE1 Fc Pichia pastorisPNGase F,

TOF/TOF (DHB). complex glycans

Native glycans removed with Endo H. New glycans attached by transglycosylation reaction

(Wei, et al., 2008c)

IgG Human in CHO cells

PNGase F, TOF (s-DHB), complex

and high-mannose glycans

To study effects of core-fucose on activity and half-life

(Kanda, et al., 2007b)

IgG Mouse in tobacco cell line

Hydrazine, TOF, glycans (2-AP)

Cells expressing human β-(1→4)-galactosyl transferase to introduce

galactose

(Fujiyama, et al.,

2007b)

IgG Human/mouse PNGase F, TOF Fc glycans terminated with GlcNAc

increase antibody resistance to papain

(Raju & Scallon, 2007)

IgG (Cetuximab) Human/mouse

PNGase F, Q-TOF (2-AA), glycans,

NP-HPLC

Structural determination, complex glycans

(Qian, et al., 2007)

IgG1 Maize, rice, CHO cells

Trypsin, TOF (CHCA),

glycopeptides

Parallel development of a HPLC-MS method for quantification. complex

and paucimannosidic glycans

(Karnoup, et al., 2007)

IgG1 Hamster/human chimera PNGase F, TOF To compare agonist activities of

various fucosylation variants (Scallon, et al., 2007a)

IgG1 Hamster/human chimera PNGase F, TOF To produce IgG with N-glycans

lacking fucose. (Kanda, et al., 2007a)

IgA1 CHO cells Hydrazine, TOF To produce IgG with N-glycanslacking fucose.

(Imai-Nishiya, et al., 2007)

IgG1/ Humanized PNGase F, TOF

(THAP), QIT-TOF, Glycopeptides

Glycosylation found not to contribute to extra IEF bands in batch of

antibody (caused by lysine clipping)

(Antes, et al., 2007)

IgG1 (2F8) Human in mice PNGase F, R-TOF

(DHB), glycans (de-Neu5Ac)

Effect of fucosylation on cytotoxicity (Peipp, et al., 2008)

Interferon-β1a Human Trypsin, R-TOF, glycopeptides

Function of N-glycans. Sialic acids found to be essential for high activity

(Dissing-Olesen, et al., 2008,

Wei, et al., 2008a)

Lactoferrin Pichia pastoris PNGase F, TOF, glycans

Combinatorial library approach to mimic mammalian glycosylation

(Choi, et al., 2008)

4



glycans. Those triantennary glycans branched on the 3-antenna(5/36) contained a prominent ion at m/z 831 whereas the6-branched isomer (50) did not. The galactose-containingbisected glycan (49), which is isomeric with the normallyoccurring triantennary glycans, produced a major ion by lossof the Gal–GlcNAc moiety from the ‘‘D’’ ion containing the6-antenna (Harvey, 2005b; Harvey et al., 2008a), the branchingmannose and the bisect. A galactose-substituted bisectedhybrid glycan (51) was also identified in the stem cell markerglycoprotein 19A suggesting that these substituted, bisectedglycans may be more common than originally thought.

N-Linked glycans from non-mammalian species. Onehundred and sixteen complex and paucimannosidic glycanshave been identified in the freshwater snail (Biomphalariaglabrata) (Lehr et al., 2007). Some carried 3-O-Me-mannose. This carbohydrate has also been reportedas terminating the antennae of high-mannose and paucimanno-sidic glycans from several other gastropods such as Limaxmaximus, Cepaea hortensis, Planorbarius corneus, Ariantaarbustorum, and Achatina fulica (Gutternigg et al., 2007a).Rapana venosa N-glycans have been found with HexNAc-(HexA)Fuc–GlcNAc-chains. TheHexA residues were stabilizedfor MALDI analysis by conversion of their COOH groups toamides using the procedure described by Sekiya, Wada, andTanaka (2005).

The human pathogen, Entamoeba histolytica, the agent thatcauses amebic dysentery, has been found to synthesizeN-glycanswith galactose and Glc–Gal chains attached directly to themannose residues of the trimannosyl-chitobiose core (52–55)(Magnelli et al., 2008). More N-glycans from Caenorhabditiselegans with highly substituted cores have been identified. ThusTakeuchi et al. (2008) have foundMan3GlcNAc2 substitutedwithmultiple Fuc, Fuc-Gal, and Fuc-Hex groups (e.g., 56–59) on both


Lysosomal acid lipase

Human in tobacco

Pronase, PNGase A, TOF (DHB),

glycans

For enzyme therapy of Wolman disease (cholesteryl ester storage disease)

(Du, et al., 2008)

Native glycans Aspergillusnudulans, A.

niger

PNGase F, TOF, glycans

Enzyme modification to induce mammalian-type glycosylation

(Kainz, et al., 2008)

Native glycans Arabidopsisthaliana TOF

Plants engineered to produce Neu5Ac. Did not affect wild type

glycan profile

(Castilho, et al., 2008)

PLAG peptide from

podoplanin

Saccharomyces cerevisiae TOF Yeast engineered to produce mucin-

like glycopeptides (Amano, et al., 2008)

Single-chain diabody (scDb) HEK 293 cells


(per-Me)

Glycosylation to improve pharmacoknetics

(Stork, et al., 2008)

Tobaccoglycans with

bisecting GlcNAc

Leaves Trypsin, TOF/TOF

(ATT), LIFT, Glycopeptides

Bisected glycans produced following introduction of GlcNAc transferase

III

(Rouwendal, et al., 2007)


methods.

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core GlcNAc residues and forming antenna in a fraction thatbound toC. elegans galectin LEC-6. The thermophilic archaeon,Pyrococcus furiosus uses a heptasaccharide HexNAc-(Pent)HexA-(Pent)Hex-Hex-HexNAc rather than the more commontrimannosylchitobiose-based glycans to attach to asparagine inthe normal Asn-Xaa-Ser(Thr) consensus sequence (Igura et al.,2008). HMW1 adhesin from Haemophilus influenzae has beenfound to have a single hexose or Hex2 attached to most of theasparagine residues in the normal N-link consensus sequence.The authors detected glucose by GC/MS but could not be certainthat this was not a contaminant (Gross et al., 2008). The usualendoglycosidase used for removing N-glycans, PNGase F, didnot hydrolyse these sugars. MALDI-TOF analysis has confirmedthat the sialic acid involved in the binding of the opportunisticpathogenic fungus, Aspergillus fumigatus, is Neu5Ac (Warwaset al., 2007).



Bi- and tri-sialylated complex glycans from the Atlanticsalmon (Salmo salar) have been shown to contain one O-acetylgroup under normal conditions. However, under stress, thenumber of acetyl groups rises so that some fish have biantennaryglycans with two di-acetylated sialic acids. Samples forMALDI-TOF analysis were permethylated, a reaction that removedthe acetyl groups. However, the compounds were eventuallydetected by CE-MS on the native glycans (Liu et al., 2008c).

i. Biopharmaceuticals. This is another rapidly growing areawith many expression systems in use for the production ofrecombinant therapeutics. Recent reviews in this area includethose on recombinant protein pharmaceuticals (Srebalus Barnes& Lim, 2007), N-glycosylation of recombinant therapeuticglycoproteins in plant systems (Balen & Krsnik-Rasol, 2007)and on methods for determining glycosylation for qualityassurance of antibody pharmaceuticals (Kamoda & Kakehi,2008).

A major concern in this area is the presence of non-humanglycans on the recombinant glycoproteins, particularly antigenicglycans such asa-galactose, Neu5Gc anda3-linked fucose on thecore of N-linked glycans. MALDI-TOF analysis has played amajor role in the identification of these sugars and for monitoringsystems where the glycosylation has been modified to reflecthuman glycosylation. For example, glycans containing abisecting GlcNAc residue have been produced in tobacco plantsfollowing introduction of human GlcNAc-transferase III (Rou-wendal et al., 2007). Monoclonal antibody samples derived fromtransgenic plants (plantibodies) such as IgG1 from maize, oftencontain significant amounts of aglycosylated variants that showdifferent functional and pharmacokinetic properties from theglycosylated proteins. A novel HPLC–UV–MS method tomeasure relative and absolute amounts of non-glycosylated, de-glycosylated, and total glycosylated protein has been developedand compared with glycopeptide profiling by MALDI MS. Theresults demonstrated that the quantitative aspect of HPLC–UVmethod was superior to MALDI MS profiling, which signifi-cantly overestimated the relative amounts of aglycosylatedspecies in the isolated glycopeptide fractions (Karnoup,Kuppannan, & Young, 2007).

Analysis of plantibody samples often requires analyticalmethods that are capable of N-glycan analysis from very smallamounts of material. To meet this demand, Seveno et al. (2008)have extracted glycoproteins from a few leaves (500mg) fromNicotiana tobacum (tobacco) or Medicago sativa (alfalfa) by aphenol/ammonium acetate procedure followed by deglycosyl-ation with PNGase A. The glycans were labeled with 2-AB andprofiled by MALDI-TOF or HPLC. Negative ion fragmentationby ESI-Q-TOF MS of the unlabeled glycans showed that theplant-derived glycans that contained a three-linked fucoseresidue at the reducing terminus could be differentiated fromthose containing a six-linked fucose by the absence of aprominent 2,4A cross-ring fragment ion from the reducing-terminal GlcNAc. Zietze, Muller, and Brecht (2008) have usedMALDI-TOF analysis to set up a system based on HPLC forstandardization and validation of an analytical scheme withrespect to specificity, linearity, repeatability, and lower limits ofdetection and quantitation for batch-consistency in N-glycosy-lation analysis of recombinantly produced glycoproteins.Following validation, defined limits for method variability couldbe calculated and differences observed in consistency analysis

could be separated into significant and incidental ones. Otherexamples of the use for MALDI MS for the analysis ofbiopharmaceuticals are listed in Table 6.

3. O-Linked Glycans

Reviews of methods for detecting proteins containingO-GlcNAcgroups (Wang & Hart, 2008) and of methods used for thedetermination ofO-linked glycosylation sites has been published(Yoshinao&Michiko, 2008). Determination of the structures andsites of attachment ofO-linked glycans is more difficult than thatfor N-glycans because of the absence of a consensus amino acidsequence, the tendency for multiple sites to cluster on a protein,the absence of a general endo-O-glycosidase for releasing theglycans and the absence of a common core structure. Added tothis, the multiple GalNAc-transferases that attach the initialGalNAc residue to serine or threonine residues have overlappingsubstrate specificities and the usual presence of a high prolinecontent inhibits successful fragmentation at all amide linkages.Wada and Tajiri (2008) have recently reviewed these problemsand summarized various approaches that can be used to obtaininformation on these intractable compounds.

A method to detect sites of O-glycosylation or phosphor-ylation called chemically targeted identification (CTID) has beenreported (Hathaway, 2007). Peptides containing phosphorylatedor glycosylated serine or threonine undergo b-elimination tointroduce a double bond. Nucleophilic addition of two-amino-ethanethiol to this bond yields aminoethylcysteine thatmakes thesites susceptible to lysine endopeptidase yielding products thatcould be analyzed by mass spectrometry.

a. Studies on O-linked glycopeptides. Takeuchi et al. (2007)used aspartic N-peptidase to obtain glycopeptides from flagellinderived from Pseudomonas syringae and showed that thepathogen contained the unique trisaccharide b-D-Quip4N(3-OH-1-oxybutyl)-2-Me-(1! 3)-a-L-Rhap-(1! 2)-a-L-Rhapattached to serine. Different pathovars contained different ratiosof D- and L-Rha.

Kobayashi et al. (2007) used three methods to determine thesites of attachment of O-glycans to murine fibulins. In the firstmethod, the sample was partially deglycosylated by successiveexoglycosidase treatment with Clostridium perfringens siali-dase, bovine kidney b-galactosaminidase and b-hexosaminidasefollowed by reduction and alkylation according to standardprotocols. The sample was then digested with trypsin and V8protease and the peptide fragments were isolated by solid-phaseextraction on a Pep-Clean C18 Spin column (Pierce) for analysisby MALDI-TOF MS. In a second approach, the protein wasdesialylated by mild acid treatment (0.1M aqueous trifluoro-acetic acid, 808C, 1 h), followed by trypsin/V8 digestion of thereduced and alkylated sample. In a third approach, the samplewas reduced and alkylated prior to trypsin and V8 treatment, butthe peptides and glycopeptides were analyzed by ESI-MSwithout cleavage of the sugars. Some of the O-glycan sites wereidentified but, unfortunately, the relative results from the threemethods were not described.

b. Release of O-linked glycans. Unlike the casewithN-glycans,there is no known non-specific endoglycosidase that can be usedto release O-glycans. In a search for new glycosidases, Iwase(2008) has investigated the activity of endo-GalNAc-ase from

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Streptomyces sp. OH-11242 but, like existing enzymes, thisenzymewas also found to be specific, only releasing Core 1-typestructures (Gal-b-(1! 3)-GalNAc).

In the absence of suitable endoglycosidases, O-glycans areusually released by b-elimination with sodium hydroxide and areducing agent to prevent base-catalyzed decomposition of theproducts. Unfortunately, this method removes the reducingterminus, thus preventing subsequent labeling reactions. Mildermethods that avoid the reductive step have, consequently, beeninvestigated. Use of hydrazine is an alternative release methodand recently Natsuka (2008) has published brief experimentaldetails of a technique suitable for small-scale work.

Another mild procedure that does not require the use of areducing agent uses ammonia to release the glycans. Althoughthe reaction was reported to produce quantitative release of O-glycans linked to serine and threonine residues in glycopeptidesand glycoproteins, a recent study (Tarelli, 2007) with humanIgA1 has found incomplete liberation of O-glycans with thisapproach. MALDI-TOF MS analysis revealed that only oneof the six glycosylated sites is susceptible to b-elimination underthese conditions. It was proposed that resistance to b-eliminationwas due to very close proximity of proline to the glycosylatedserine or threonine residues. Preliminary results using0.1M NaOH instead of ammonia indicated that there was alsoselective hydrolysis of peptide bonds. The author commentedthat the findings might have implications for similarlyO-glycosylated peptides and proteins and possibly for otherchemical methods that are used to carry out b-eliminations ofO-glycans.

In another mild method for releasing O-glycans, Kisiel,Radziejewska, and Gindzienski (2008) have used a 50% aqueoushydrazine solution containing 0.2M triethylamine. The mixturewas heated at 458C and an optimum time of 48 hrwas determinedfor release of O-glycans from pig gastric mucin. Recoveredglycans were comparable with those released by b-eliminationbut the hydrazine releasemethod had the advantage of leaving thereducing termini of the glycans intact. One drawback, however,was the need to re-N-acetylate the amino-sugars.

Because of the long reaction times that usually accompanyb-elimination reactions, Matsuno, Yamada, and Kakehi (2008)have developed an in-line flow apparatus, called the AutoGly-coCutter, employing LiOH at 708C for releasing O-linkedglycans at high temperature (608C) but with short reaction timesfollowed by rapid neutralization to limit glycan degradation.Consequently, the reductive step could be eliminated, preservingthe reducing terminus. Released glycans were labeled with 2-AAand analyzed by MALDI-TOF mass spectrometry. Glycanrelease was claimed to take only 3min (Matsuno et al., 2007).An extension to the system incorporates a roboticMALDI spotter(AccuSpot) that sampled a small amount of the reaction mixture.Using MALDI-TOF analysis, mucin-type glycans from bovinesubmaxillary mucin, bovine fetuin, porcine stomach mucin,and human colostrum immunoglobulin A could be analyzed inonly 3 hr.

Williams, Saggese, and Muddiman (2008a) have optimizedvarious parameters for sample clean-up prior to glycan release byb-elimination. They note that, for lectin chromatography forglycoprotein enrichment prior to b-elimination, gains in samplecleanup did not offset sample loss because of additionalchromatography steps. Samples were exhaustively digested withpronase to remove protein contamination and it was found that

the optimum reaction time was 4 hr with 5mg/mL of pronase.Deactivation of the pronase at the end of the digestion improvedthe quality of the data. Drop dialysis for 2 hr was used for furtherclean-up, longer times resulted in glycan loss. The optimizedtechnique was used to study O-glycans in ovarian cancer. In afurther paper (Williams et al., 2008b), this group have optimizedthe mass spectrometric side of the analysis and have shown, forexample, that higher ion abundance can be obtained if the DHBmatrix spot is actively dried with a stream of air rather than beingallowed to dry under ambient conditions.

A single-step method to isolate sulfated O-linked glycansbased on the exclusion of sulfated molecules from strong cationexchange resin has been reported by Garenaux et al. (2008). O-glycans were released from frog egg jelly coats and humantracheobronchial mucins by b-elimination and purified by a two-step fractionation using a strong cation exchange column. Thefirst column (Dowex 50x2) was used to remove peptides andthe eluted neutral and sulfated carbohydrates were fractionatedon the second column. The use of lectins to isolate O-glycansfound in egg jelly coats of the frog Xenopus laevis has beendescribed by Kirmiz, Chu, and Lebrilla (2007a).

Detailed protocols for N- and O-linked glycan release andanalysis, based on the well-established methods of PNGase Frelease of N-glycans and b-elimination of O-glycans have beenpublished (Morelle & Michalski, 2007). Glycan analysis wasperformed by exoglycosidase digestion and mass spectrometry(ESI, MALDI-TOF, and GC/MS) on permethylated samples.

c. Applications of MALDI to the structural determinationof O-linked glycans. Most applications are summarized inTable 7 (specific glycoproteins) and Table 8 (tissues and wholeorganisms). Only studies reporting more unusual or new glycansage included here.

Most O-linked glycans are attached to serine or threoninebut Jensen et al. (2007) have detected small O-linked glycans(Gal–Glc) attached to hydroxylysine residues from humanplasma mannose-binding lectin. Glycoprotein I of herpessimplex virus type 1 has been found to contain a uniquepolymorphic tandem-repeated mucin region (STPSTTTSTP-STTT) that acts as a substrate for several GalNAc transferasesthat form a polymorphic mucin region not previously describedfor the virus (Norberg et al., 2007). The first analyses of theO-glycans of microsproidia have been reported (Taupin et al.,2007). Both Encephalitozoon cuniculi andAntonospora locustaecontained linear manno-oligosaccharides with chain lengths ofup to eight a-(1! 2)-linked mannose residues, similar to thosereported from fungi such as Candida albicans. O-glycans fromthe 114-4D12-binding glycoprotein from Schistosoma mansonisoluble egg antigen have been shown to contain O-glycanswith several fucose chains (e.g., 60), an epitope not yet seen inN-glycans (Robijn et al., 2007b). The free glycans weredetectable in the urine of infected humans (Robijn et al.,2007a). An unusual monosaccharide attached to Ser-63 of PilE,the major subunit of type IV pilin from Neisseria meningitidis,the causal agent of cerebrospinal meningitis, has been identifiedas a hexose containing an N-acetylamido group at C2 and anN-glyceramido group at C4 (61). MALDI-TOF analysis of theglycoprotein gave a mass of 17,496Da (�5Da) which wasreduced to 17,209� 11Da when serine 63 was mutated toalanine, giving a mass for the glycan of approximately 271. Thestructure was determined by ESI MS/MS following cleavage of



TABLE 7. Use of MALDI MS for examination of O-glycans from specific glycopeptides

Glycoprotein Type Methods1 Notes Reference

β-Actin Xenopus laevisOocyte

Trypsin, TOF/TOF (CHCA), ESI

Effect of O-GlcNAc formation on cell division

(Dehennaut, et al., 2008)

Ag43α Escherichia coli Trypsin, TOF (DHB), LC-MS/MS

Structural determination. Effect of glycosylation

(Knudsen, et al., 2008)

Akt1 Mouse pancreatic cells

Trypsin, TOF/TOF (CHCA), glycopeptides

O-GlcNAc modulation at Ser473 correlates with

apoptosis

(Kang, et al., 2008a)

AMACO (VWA2 Protein)

Recombinent β-elimination, TOF

(DHB), glycans (per-Me), ESI, GC/MS

O-Glc and O-Fuc in close proximity on first epidermal

growth factor repeat of AMACO. Overlapping

consensus sequence

(Gebauer, et al., 2008)

Bone morphogenetic

protein-15 (BMP-15)

Human in HEK293 cells

TOF (sinapinic), LC-MS/MS, Intact glycoprotein

Structural determination (NeuAc2HexHexNAc)

(Saito, et al., 2008)

CD52 Human

β-Elimination, TOF, TOF/TOF, (DHB, CHCA)

ESI-MS/MS, glycans (per-Me)

Structural determination (Parry, et al., 2007b)

Coagulationfactor VII Human plasma

Trypsin, Asp-N, TOF (DHB, THAP, CHCA),

LC-ESI-MS, glycopeptides

Structural determination. Contained glucose and

xylose

(Fenaille, et al., 2008)

Carbonic anhydrase IX

Human in murine NS0

myeloma cells

Trypsin, L-, R-TOF (DHB, THAP/NH4 cit),

glycopeptides Structural determination (Hilvo, et al.,

2008)

α-Dystroglycan, mucin-like

domain Synthetic TFA, TOF (DHB),

glycopeptides

Preferred amino acid sequence for protein

mannosylation

(Manya, et al., 2007)

Egg-secreted protein

Schistosoma mansoni (eggs and cercarial

secretion)

β-Elimination, TOF, TOF/TOF (DHB), ESI-

MS/MS, glycans (per-Me) Structural determination (Jang-Lee, et

al., 2007)

Fap1 (serine-rich adhesin)

Streptococcus parasanguinis

β-elimination, TOF (DHB), GC/MS, glycans

(per-Me)

Interaction between two glycosyltransferases

required for glycosylation

(Bu, et al., 2008)

Fibulin Mouse β-Elimination, TOF

(DHB), ESI, glycans (Per-Me)

Biochemical characterization of five

fibulins

(Kobayashi, et al., 2007)

Flagellin Pseudomonas syringae

Aspartic N-peptidase,TOF, QIT-TOF (DHB),

ESI, HPLC, NMR, glycopeptides

Structural determination. Pathovars contain D- and L-Rha in different proportions

(Takeuchi, et al., 2007)

Flagellin Campylobacterjujuni

Trypsin, FT-ICR (DHB), glycopeptides

Maf4 gene produces novel pseudominic acid

glycoforms

(van Alphen, et al., 2008)

IgG 114-4D12-binding

glycoprotein

Schistosoma mansoni

β-Elimination, TOF/TOF (DHB) glycans (2-AB)

High fucose chains identified

(Robijn, et al., 2007b)

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Glycopeptides program to predict O-sites al., 2007) Mannose

binding lectin Human plasma TOF, TOF/TOF (CHCA) Structural determination. Glycans on hydroxy-lysine

(Jensen, et al., 2007)

Microcystin-related protein

Microcystisaeruginosa PCC

7806

Asp-N, TOF, PSD, glycopeptides Structural determination (Zilliges, et al.,

2008)

MUC1 Human synthetic glycopeptides

Glycopeptides, R-TOF (DHB), LC-MS/MS Glycans inhibit proteolysis (Ninkovic &

Hanisch, 2007)

Mucin Rat sublingual β-elimination, TOF, TOF/TOF (DHB),

glycans (per-Me), GC/MS

Structural determination. (Core III, Core IV)

(Yu, et al., 2008a)

Mucin Pig gastric β-Elimination, TOF (DHB), glycans, NMR

Determination of binding epitope to monoclonal

antibody PGM34 as Fuc-α 1→2Gal β1→4GlcNAc-

(6SO3H) β-

(Tsubokawa, et al., 2007)

Mucin Pig gastric β-Elimination, TOF (DHB), ESI

Molecular structure and rheological properties

(Yakubov, et al., 2007)

Mucin Jellyfish (5 species) TOF (IAA) Structural determination (Masuda, et

al., 2007)

Notch receptor Drosophila

expressed in S2 cells

Trypsin, TOF, TOF/TOF (sinapinic for protein, CHCA for peptides)

1st report of O-GlcNAc in extracellular environment

(Matsuura, et al., 2008)

Osteopontin Mouse Trypsin, L-TOF, glycopeptides

Post-translational modifications affect

adhesion

(Christensen, et al., 2007)

Pilin (Type IV) Neisseriameningitidis

ESI, in source, TOF (sinapinic), FT-ICR, Q-

TOF (ESI)

Structural determination, Unusual hexose, see text.

(Chamot-Rooke, et al.,

2007)

Podoplanin Human

(glioblastoma cell line)

β-Elimination, QIT-TOF (DHB), glycans (per-Me)

Structural determination. Sialylation critical for

podoplanin-induced platelet aggregation

(Kaneko, et al., 2007)

Pregnancy-associated

glycoproteins Pregnant cow

β-elimination, TOF/TOF (THAP/NH4-Cit -ve,

DHB, +ve), glycans (per-Me)

Structural determination. (Core I, Core II)

(Klisch, et al., 2008)

Protein C inhibitor Human plasma

β-elimination, R-TOF (DHB), TOF/TOF (DHB, CHCA), glycans (per-Me)

Structural determination. (Core 1)

(Sun, et al., 2008b)

Vitamin D binding protein Human plasma

Arg-C, TOF/TOF (THAP, NH -Cit, CHCA),

glycopeptides Structural determination (Borges, et al.,

2008) 4

Insulin receptor Human Asp-N, Trypsin, TOF, Shows failure of web (Sparrow, et


methods.



the glycan in the ion source of a Q-TOF instrument (Chamot-Rooke et al., 2007).

TABLE 8. Use of MALDI MS for examination of O-glycans from intact organisms or tissues

Organism Type Methods1 Notes Reference

Antonospora locusta Spores

β-Elimination, R-TOF (DHB), glycans (Free,

Per-Me)

Structural determination. First microspora O-glycans.

(Mannose1-8)

(Taupin, et al., 2007)

Caenorhabditiselegans mutant Total extract β-Elimination, R-TOF


Bre-1 Mutant produces glycans deficient in fucose.

Composition only

(Barrows, et al., 2007)

Encephalitozooncuniculi Spores

β-Elimination, R-TOF (DHB), glycans (Free,

Per-Me)

Structural determination. First microspora O-glycans.

(Mannose1-8)

(Taupin, et al., 2007)

Human Ovarian cyst β-Elimination, TOF, TOF/TOF, Q-TOF


Structural determination (Core I and core II, sLex, sLea, Lea)

(Wu, et al., 2007a)

Human Dendriticcells

β-Elimination, TOF, TOF/TOF (DHB), glycans (Per-Me)

Core I up-regulated during maturation, Core II down

(Bax, et al., 2007)

Human Dendriticcells

β-Elimination, TOF, TOF/TOF (DHB), glycans (per-Me)

Sialyl-Lewisx is regulated during differentiation and

maturation. (Core I, Core II)

(Julien, et al., 2007)

Mouse Brain β-Elimination, TOF

(DHB), TOF-TOF (DHB, CHCA), glycans (per-Me)

Combination with method involving ceramide glycanase release for glycosphingolipids

(Parry, et al., 2007a)

Mouse Intestinal mucus

β-Elimination (NH ), TOF (DHB), HPLC,

glycans (2-AB)

Mice lacking Core III glycans susceptible to colonic disease

(An, et al., 2007)

Mouse Endothelial cells

NH4-based β-elimination, TOF, glycans (2-AB)

O-glycans control separation of blood and lymphatic vessels

during embryonic and postnatal development,

(Fu, et al., 2008a)

Oryctolagus cuniculus (rabbit,

New Zealand white)

Erythrocytes β-Elimination, TOF

(DHB), TOF-TOF (DHB, CHCA), glycans (per-Me)

Structural determination. To investigate why mouse sperm binds to rabbit erythrocytes

(Sutton-Smith, et al.,

2007)

Rhesus monkey Gastricbiopsies

β-Elimination, FT-ICR (DHB,+ve, THAP,-ve),

glycans, ESI

Fewer oligosaccharides from monkeys with Helicobacter

pylori infection

(Cooke, et al., 2007)

3


methods.

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Glycosaminoglycans (GAGs). Amajor problem associatedwithMALDI analysis of these compounds is loss of sulfate. Thisloss has been minimized by forming cesium salts and use of theliquid matrix, bis-1,1,3,3-tetramethylguanidinium (12)-CHCA(Laremore & Lin, 2007) The series of alkali metals, lithium,sodium, potassium, rubidium, and cesium as their chlorides orhydroxides were added to the MALDI matrix and tests werecarried out with sucrose octasulfate. The larger alkali metalsproduced little or no fragmentation allowing the sulfatedcarbohydrate to be observed as the octa-rubidium or octa-cesiumsalt in both positive and negative mode with negligible loss ofsulfate. Cesium was the preferred metal because of itsmonoisotopic nature. A further advantage of the cesium saltswas that they moved the mass of the disaccharides derived fromhyaluronan away from the matrix region.

Two matrices have been investigated by Tissot et al. (2007)for MALDI-TOF and MALDI-TOF/TOF analysis of GAG-derived di-, tetra-, hexa- and deca-oligosaccharides carryingfrom 2 to 13 sulfate groups. The first, an ionic liquid, 1-ethylimidazolium (14) a-cyano-4-hydroxycinnamate (ImCHCA),produced very little loss of sulfate, a property attributed by theauthors, to a high sodium content that stabilized the sulfates bysalt formation. By contrast, the second matrix, a crystallinebinary mixture of CHCA and 1-methylimidazole (62) producedconsiderable desulfation but was said to facilitate the determi-nation of the carbohydrate backbone and the position of theN-acetyl residues.

The ion pairing reagent, (Arg-Gly)15withDHBas thematrixhas been used to determine the structures of octa- anddecasaccharides derived from hydrolysis of chondroitin sulfateE from squid cartilage. The building blocks were determined tobe A- [GlcAb1-3GalNAc(4S)], C- [GlcAb1-3GalNAc(6S)], andE- [GlcAb1-3GalNAc(4S,6S)] units, where 4S and 6S represent4-O- and 6-O-sulfate, respectively. Among the 11 differentoctasaccharide sequences, eight were novel, while all of the sixdecasaccharide sequences were novel. This is the first report ofthe sequencing of CS oligosaccharides with chain lengths longerthan eight units (Deepa et al., 2007b). The same MALDItechnique with (Arg-Gly)15 to neutralize the sulfates was alsoused by Pothacharoen et al. (2007) to identify the octasaccharidesequences recognized by the monoclonal antibody WF6. In a

further report, 12 octasaccharide fractions have been obtainedfrom chondroitin sulfate C from shark cartilage after hyalur-onidase digestion and their sugar and sulfate compositions havealso been assigned using the (Arg-Gly)15 complexation method.The sequences were determined at low picomole amounts by acombination of enzymatic digestions and HPLC. Twenty fourdifferent sequences, of which nineteen were novel, wereidentified (Deepa et al., 2007a).

The unsulfated hyaluronan (((!3)-b-D-GlcNAcp-(1! 4)-b-D-GlcAp-(1!))n). In a comparative study of sodium salts,organoammonium salts andmethyl esters for quantitative studiesof hyaluronan oligosaccharides (2–32-mer), the methyl estersgave the best results. Spectra of the sodium salts containedmultiple peaks from several matrices but the methyl estersproduced single peaks from each oligomer with CHCA as thematrix. Methyl ester formation was achieved with trimethylsilyldiazomethane (63) (Sakai et al., 2007b).

Further studies on the application of MALDI massspectrometry to the analysis of GAGs are listed in Table 9.

4. Glycosylphosphatidylinositol (GPI) Anchors

The structure of the (GPI) anchor of the Trypanosoma bruceitransferrin receptor has been characterized and found to containthe highest numbers of hexose, presumably galactose residues ofany GPI anchor yet characterized (Mehlert & Ferguson, 2007).

F. Glycated Proteins (Non-Enzymatic Attachmentof Sugars)

A review of age-related protein modifications, includingglycation and detection bymass spectrometry has been published(Soskic, Groebe, & Schrattenholz, 2008). Gontarev et al. (2007)have developed a method for extraction and purification ofglycated proteins using phenylboronic acid attachment. Agarosehydrogel was first used to create a high-capacity affinity layer onthe modified aluminium surface of a MALDI target. 3-Amino-phenylboronic acid (5/42) was then bound via cyanogen bromideactivation as a ligand for affinity sorption of glycated proteins,

TABLE 9. Use of MALDI MS for examination of glycosaminoglycans and related compounds

Carbohydrate Species Methods1 Notes Reference

GAG chain from bikunin Commercial

TOF (CHCA), ESI-FTICR,

NMR

Structural determination. MALDI mainly for protein and glycoprotein

measurements (Chi, et al., 2008)

Heparan sulfate oligosaccharides

Porcinemucosa

(commercial) TOF

For studies with interactions with platelet endothelial cell adhesion

molecule 1 (PECAM-1)

(Coombe, et al., 2008)

1Format (not all items present): MALDI method (matrix), other methods.



followed by their direct detection by MALDI MS. Efficientbinding of glycated proteins but only low, non-specific binding ofnon-glycated proteins was demonstrated.

Lapolla et al. (2007a) have compared DHB and CHCA foranalysis of glycated peptides and found that DHB detected themost compounds even though stronger signals were produced byCHCA.Use of an off-line LC-MALDI system allowed evenmorepeptides to be detected. In a study with human serum albumin(HSA) they found that, even in the presence of high concen-trations of glucose, not all of the possible reactive sites (lysine)became glycated. MS/MS spectra of the glycated peptidesshowed diagnostic ions 120 and 162 mass units below that of themolecular ions, formed by loss of the sugar. The same group(Lapolla et al., 2007b) have detected glycation products of humanglobins from nephropathic patients by MALDI-TOF. Althoughnot all products were identified, principal component analysishighlighted several products that were involved in carbonylstress. Detection of glycation in malt samples has been achievedby use of direct examination with anion-exchange chromatog-raphy on short monolithic columns followed by MALDI-TOFanalysis of the glycated proteins (Bobalova & Chmelik, 2007).Nonspecific lipid transfer protein (LTP1) was proposed as aglycation marker during malting.

A method for separating glycated peptides from their non-glycated counterparts by reversed-phase HPLC using ion pairingwith heptafluorobutyric acid (HFBA) (C3F7COOH) has beenreported by Frolov and Hoffmann (2008). HFBA produced betterseparations than the smaller TFA by increasing the retention ofthe unmodified peptide. For MALDI-TOF or ESI analysis of theseparated glycated peptides, it was advantageous to break the ionpair with a weak volatile acid.

Most proteins are too large for individual glycated species tobe observed by MALDI-TOF. However, globin, (15 kDa) a by-product of the meat industry, has been glycated with glucose,fructose (1/16) and psicose (64) and the modification was shownto improve the rheological properties of low sodium wheatdough. The numbers of sugar molecules attached to the globin(1 and 2 for Psi, 1–3 for Fru and 1–8 for Glc) were clearlyresolved by MALDI-TOF from sinapinic acid (Innun et al.,2007). Other examples are listed in Table 10.

G. Peptidoglycans

A nonsulfated chondroitin proteoglycan has been found in thedried saliva ofCollocalia swiftlets (edible birds nest). The linkerregion hexasaccharidewas identified asHexA-a-(1–3)-GalNAc-b-(1–4)-GlcA-b-(1–3)-Gal-b-(1–3)-Gal-b-(1–4)-Xyl by acombination of HPLC (2-AB derivatives) MALDI-TOF MSand by comparison with an authentic standard (reducing endtetrasaccharide; Nakagawa et al., 2007). Other examples arelisted in Table 11.

H. Glycolipids

Recent work on the molecular structure of endotoxins fromGram-negative marine bacteria has been reviewed (Leone et al.,2007a).

1. Lipopolysaccharides (LPS)

The large lipopolysaccharides from bacteria are usually split intosegments, lipid A, core, O-chain, by taking advantage of bondsthat are labile when the molecules are heated with dilute acid,as nicely illustrated by D’Haeze et al. (2007) for the LPS fromRhizobium etli (65) (see Table 12 for further details). LPSextracted by the traditional phenol/water method is usuallycontaminated with various compounds such as phospholipids,proteins, nucleic acids, capsular polysaccharides, peptidoglycanfragments, and lipoproteins. Tirsoaga et al. (2007b) haveexamined several methods for removal of these contaminantsbased on heat treatment, acid-solvent treatment and detergent-based hydrolysis. Of the three methods, the acid treatmentwas preferred and gave greatly improved MALDI-TOFperformance.

a. Lipid A. A new rapid method for preparing lipid A involvesfirst ammonium isobutyric hydrolysis of freeze-dried cells toliberate the intact molecule and then incubation with either 35%ammonium hydroxide or 41% methylamine (Tirsoaga et al.,2007a). The first reaction causes deacylation of the GlcNAcresidues while the second also cleaves the lipid chains from thehydroxy fatty acids that form the N-acyl groups. Analysis of theproducts of this technique by MALDI-TOF of lipid A isolatedfrom Bordetella bronchisertica showed that the two phosphategroups carried hitherto unreported HexN residues.

The MS/MS spectra of lipid A isolated from several speciesof bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa,Salmonella enterica, and various strains of Yersinia) often showabundant pyrophosphate anions. Pyrophosphate [H3P2O7]

� and/or [HP2O6]

� anions are dominant fragments from diphosphory-lated lipidA anions regardless of the ionizationmode (MALDI orESI ionization), excitation mode (collisional activation orinfrared photoexcitation), or mass analyzer (TOF/TOF, tandemquadrupole, FT-ICR). In order to determine if these ionsoriginate from Lipid A-attached pyrophosphate, fragmentationof model lipids such as phosphate, pyrophosphate, and hexosediphosphates confirmed that pyrophosphate fragments wereformed abundantly only in the presence of an intact pyrophos-phate group in the analyte molecule. They were not due tointramolecular rearrangement upon ionization, ion-moleculereactions, or rearrangement following activation (Jones et al.,2008).

b. Core oligosaccharide. Analysis of the complex core ofRhizobium rubi LPS initially gave inconclusive results whenexamined by MALDI-TOF MS but, after deacylation withammonia, a good spectrum was obtained from THAP using thethin-layer procedure. The samplewas first suspended in amixtureof methanol/water (1:1) that contained 5mM EDTA, thenconverted into the ammonium form with cation-exchange beadsand was finally deposited together with the same volume of20mM dibasic ammonium citrate, on the matrix film that wasalready spotted on the MALDI plate (Gargiulo et al., 2008).

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TABLE 10. Use of MALDI to study glycated proteins

Protein/amino acid Sugar Methods1 Notes Reference

Albumin (bovine serum) Glc TOF

Use of bead-reconstituted membrane proteins to measure

AGE binding

(Schmitt, et al., 2007)

Albumin (bovine serum) Glc L-TOF

Highly modified but not mildly modified BSA binds to

scavenger receptors

(Nagai, et al., 2007)

Albumin (bovine serum) AGEs L-TOF (sinapinic,

DHB)

Glycation shown not to modify BSA-induced reduction of rat

aortic relaxation

(Rubio-Ruiz, et al., 2008)

Albumin (bovine serum) Glc, Gal, lactose TOF (CHCA)

Incubation at 60oC for different times and glycated products

tested for recognition by lectins

(Ledesma-Osuna, et al.,

2008)

Albumin (human serum)

AGEs from Me-glyoxal TOF (CHCA)

Cytotoxicity of AGEs in human micro- and astroglial cell lines depends on degree of glycation

(Bigl, et al., 2008)

Albumin (human serum) Glc

R-TOF (DHB, CHCA), LC-MALDI,

Glycopeptides

Regardless of high glucose concentration, glycation does not

go to completion.

(Lapolla, et al., 2007a)

Albumin (human serum)

minimally glycated

- TOF (DHB/CHCA) Characterization of glycation

products following trypsin, Lys-C or Glu-C digestion

(Wa, et al., 2007)

Albumin (human and

bovine serum)

Glucose, methyl glyoxal

TOF/TOF (sinapinic), ESI

Glycoxidized albumin induces oxidative stress on human

monocytes

(Rondeau, et al., 2008)

ApoA-1

From human plasma by 2-D

gel electrophoresis

TOF (CHCA)

Plasma from diabetic and nephropathic patients contained increased amounts of glycated

and unglycated ApoA-1

(Lapolla, et al., 2008b)

Creatine kinase, carbonic

anhydrate III, β-enolase, actin

- TOF

Shows that metabolic proteins, particularly β-enolase contain increased AGE modifications

with age

(Snow, et al., 2007)

Globin Glc, Fru, Psi TOF (sinapinic) Effect of glycation on

rheological properties of bread dough

(Innun, et al., 2007)

Humanized antibody, α-D-Glc TOF/TOF (CHCA),

LC-ESI-MS/MS High level of glycation found at

Lys 49 on the light chain (Zhang, et al.,

2008a)

IgG Sucrose MALDI Effect of temperature on protein glycation

(Gadgil, et al., 2007)

α- and βlactoglobulin

Lactose TOF (DHAP, Di-

NH4-citrate, CHCA, Di-NH4-citrate)

Site-specific formation of Maillard, oxidation, and

condensation products from whey proteins

(Meltretter, et al., 2007)

α-Lacalbumin, β-lactoglobulin

Lactose TOF, ESI Detection of glycated and oxidized products in whey

proteins from milk

(Meltretter & Pischetsrieder,

2008)

α- and β-lactoglobulin

and IgG

Xylobiose maltose TOF (sinapinic)

Nutrititive properties of whey improved by glycation.

Xylobiose reacts faster than maltose

(Yajima, et al., 2007)

-



c. O-Chain. Most O-chains consist of repeating units ofrelatively short oligosaccharides that can be accessed by acidhydrolysis. The MALDI-TOF-MS spectrum of the wholeSalmonella. Dakar O-polysaccharide has shown several ions upto m/z 9,000, with differences of about 860Da between their m/zvalues confirming the mass of the repeating pentasaccharide.The uniqueO-chain (!3)-b-D-FucpNAc4N-(1!)1)-D-Rib-ol-5-P-(O! 1)-D-Rib-ol-5-P-(O!) with random acetylation fromProteous vulgaris TG 276-1 has been depolymerised byhydrolysis at the phosphate linkages with 48% aqueous HF forMALDI-TOF analysis. The unique nature of the carbohydrateprompted the authors to reclassify the bacterium into a newsubgroup.

2. Glycosphingolipids

Analysis of sphingo- and glycosphingo-lipids has been reviewed(Merrill et al., 2007; Sullards et al., 2007).

a. Matrices. THAP has been shown to be a versatile matrixfor glycosphingolipid analysis in a study comparing ninematrices. Its high salt tolerance was compatible with dopingwith sodiumor lithiumacetate to promote cationization (Stubiger& Belgacem, 2007). On the other hand, Garcia, Callewaert, andBorsig (2007) have, by comparing most commonly used

matrices, reaffirmed thatATT is one of the best. Their preparationconsisted of ATT containing 10mM NH4-citrate. This matrixproduced few matrix ions in the low mass region and issufficiently ‘‘cool’’ so as not to catalyze significant losses ofsulfate or sialic acid. Cha and Yeung (2007) have comparedcolloidal graphite-assisted laser ionization MS with high andintermediate pressure vacuum MALDI (DHB matrix) foranalysis of cerebrosides in total brain extracts. The graphite-assisted technique dramatically increased the signals from thecerebrosides at the expense of that from phosphatidylcholine,normally the most abundant signal in these spectra. The effectwas more significant in the high than the intermediate vacuumspectra.

Zarei et al. (2008b) have compared several methods foranalysis of methyl-esterified gangliosides and have achieved thebest results with ATT/diammonium citrate as the matrix in anorthogonal-TOF instrument with nitrogen cooling gas (1mBar)to reduce loss of sialic acids. Themethodwas used to characterizegangliosides from mouse brain. The same group (Zarei et al.,2008a) have developed an LC-MS system using a MALDIspotting device with DHB (neutral) and the ATT/diammoniumcitrate matrix for gangliosides and used it to examine neutralGSLs purified fromhuman erythrocytes and amonosialoganglio-side mixture expressed by the murine MDAY-D2 cell line.


β-Lactoglobulin Galacto-oligosaccharides

L-TOF (sinapinic), HPLC

Study of reaction of prebioticgalacto-oligosaccharides with

protein in connection with human nutrition

(Luz Sanz, et al., 2007)

β-Lactoglobulin Galactose from

galacto-oligosaccharides

TOF/TOF (CHCA), LC-ESI-MS/MS

To characterise glycated peptides produced by in vitro

gastrointestinal digestion

(Moreno, et al., 2008)

β-Lactoglobulin (bovine)

Galactose tagatose

TOF (sinapinic), SEC, HPLC

To investigate relative reactivities of aldose (Gal) or

ketose (Tag). Gal more reactive than Tag

(Corzo-Martínez, et

al., 2008)

Lipoprotein ApoA-1 Glc TOF (CHCA)

Overexpression of glycated and un-glycated protein in diabetic and

nephropathic patients

(Lapolla, et al., 2008a)

Milk proteins Lactose R-TOF/TOF (CHCA) To study the effect of heat-

induced lactosylation on proteolysis (Dairy industry)

(Dalsgaard, et al., 2007)

Peptides D-Glc TOF (CHCA) Separation of Amadori peptides from unmodified analogs by ion-pairing RP-HPLC with HFBA.

(Frolov & Hoffmann,

2008)

Ribonuclease A Glc TOF (CHCA)

First documentation that both intra- and inter-molecular cross-links form in glucose-incubated

proteins.

(Dai, et al., 2008)

Various Glucose TOF (CHCA, sinapinic), ESI

Glycation increases with temperature and Glc conc. Little

affected by pH.

(Lee, et al., 2007a)

Yeast proteins Me-glyoxal TOF (CHCA) Effect of glycation on yeast

enzymes. Enolase was major target

(Gomes, et al., 2008a)

(Gomes, et al., 2008b)

1Format (not all items present): MALDI method (matrix), compounds run (derivative), other methods.

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TABLE 11. Use of MALDI MS for investigations of bacterial peptidoglycans

Species Peptidoglycan Methods1 Notes Reference

Bacillus anthracis BcpAMH6 anchor peptides

R-TOF/TOF(CHCA) Structural determination (Budzik, et al.,

2008)

Escherichia coli Peptidoglycan TOF C-terminal domain of Escherichia coli YfhD shown to function as a

lytic transglycosylase

(Scheurwater & Clarke, 2008)

Lactococcus lactis Peptidoglycan TOF, PSD

Enzymic N-acetylglucosamine deacetylation protects

peptidoglycans from hydrolysis by the major autolysin

(Meyrand, et al., 2007)

1Format (not all items present): MALDI method (matrix).



TABLE 12. Use of MALDI MS for examination of bacterial glycolipids (Lipid A-linked)

Species Type Methods1 Notes Reference Aeromonas

hydrophila AH-3 (serotype O34)

LPS TOF (DHB), GC/MS

Effect of epimerase enzymes on virulence

(Canals, et al., 2007)

Acinetobacterradioresistens S13 Lipid A TOF (THAP),

GC/MS

Structural determination. First report of lipid A structure from

Acinetobacter

(Leone, et al., 2007b)

Alteromonas addita LPS L-TOF

(THAP), GLC, GC/MS, NMR

Structural determination. Terminal glucose residue attached via a

phosphate group.

(Leone, et al., 2007c)

Azospirillum lipoferum

(rhizobacterium)Lipid A TOF (DHB),

GC/MS

Structural determination. Unusual in having no phosphate groups. -linked D-galacturonic acid at C1

(Choma & Komaniecka,

2008) Bordetella

bronchiseptica,strain 4650

Lipid A L-TOF (DHB) Method for recovering lipid A.

Phosphate groups substituted with HexN

(Tirsoaga, et al., 2007a)

Bordetella hinzii O-chain and core

TOF (2,4-DHB), NMR Structural determination (Vinogradov,

2007) Bordetella pertussis

Tohama I and B.bronchiseptica strain

4650

Lipid A L-TOF (DHB), PSD

Structural determination. Glucosamine found as a substituent

of both phosphate groups. Not previously described in Bordetella

(Marr, et al., 2008)

Burkholderia cenocepacia ET-12 LPS TOF, NMR Structural determination (Silipo, et al.,

2007)

Burkholderia mallei Lipid A TOF (DHB), R-TOF/TOF Structural determination (Brett, et al.,

2007)

Burkholderia multivorans LPS

TOF, R-TOF/TOF (THAP),

GC/MS, NMR

Structural determination. Proinflammatory activity and

differences between strain colonizing pre- and post-transplanted lungs

(Ieranò, et al., 2008)

Burkholderia pseudomallei Lipid A TOF (CMBT) Activation of Toll-like receptors (West, et al.,

2008) Campylobacter

jejuni LOS TOF (DHB), GC/MS

Structural determination. Interaction with cholera toxin B subunit

(Usuki, et al., 2007)

Campylobacterjejuni CG8486 (serotype HS:4)

Capsular poly-

saccharide

TOF(sinapinic),

GC/MS, NMR

Structural determination. Identification of 6-deoxy-D-ido-Hepp

(Chen, et al., 2008e)

Citrobacter species Lipid A L-TOF (DHB) Method for recovering lipid A. Three species had identical lipid

(Tirsoaga, et al., 2007a)

Escherichia coli(various strains) Lipid A TOF (DHB)

Affect on Lipid A structure and sensitivity to cationic antimicrobial

peptides and vancomycin

(Lamarche, et al., 2008)

Escherichia coliand Shigella flexneri

2a Lipid A TOF (ATT) Combination of acylation state and

host genetics affects use as vaccine (Rallabhandi, et

al., 2008)

Escherichia coli Lipid A/LPS R-TOF (DHB) Analysis of proteins required for

transport of LPS to outer membrane. Conjugation with colanic acid

(Sperandeo, et al., 2008)

Francisella novicida Lipid A TOF/TOF (CMBT)

Mutants without one or both of the attached GalpN or Manp studied with

respect to inate immune response

(Kanistanon, et al., 2008)

Francisellatularensis Lipid A LTQ, TOF

(CMBT) Identification of LpxL, a late

acyltransferase (McLendon, et

al., 2007)

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Francisellatularensis Core

TOF/TOF (THAP/ NH4Cit/

nitrocellulose), GC/MS

Genetic modification to remove O-chain removes virulence and

compromises protective immunity in mice

(Li, et al., 2007d)

Francisellatularensis subsp.

novicidaLipid A TOF

Structural heterogeneity and environmentally regulated

remodeling

(Shaffer, et al., 2007)

Francisellatularensis,

F. novicida,F. philomiragia

Lipid A Ion trap, (CMBT), MSn

Structural determination. GalN or hexose on single phosphate.

(Schilling, et al., 2007)

Francisella spp. Lipid A, LPS MALDI

MALDI-TOF used to show no difference in lipid A and LPS in a

mutant

(Mohapatra, et al., 2007)

Haemophilus influenzae 2019

DeacylatedLOS

Ion trap (DHB)

Characterization of the Neu5Ac binding site of the extracytoplasmic

solute receptor (SiaP)

(Johnston, et al., 2008)

Fusobacteriumnucleatum sp.

nucleatum Lipid A TOF/TOF

(DHB)

Comparison with lipid A from E.coli. sCD14 shown to discriminate

slight structural differences

(Asai, et al., 2007)

Haemophilus ducreyi LOS MALDI-LTQ Structural determination,

identification of genes (Post, et al.,

2007)

Haemophilus ducreyi LOS

MALDI-LTQ (DHB), Glycans

Structural determination. Similar structures from several strains.

(Post & Gibson, 2007)

Haemophilus influenzae Total L-ion trap

(DHB) Regulation of sialic acid transport

from host (Johnston, et al.,

2007)

Helicobacter pylori Lipid A TOF (ATT) Biochemistry of acylation and deacylation

(Stead, et al., 2008)

Klebsiellapneumoniae Core-OS TOF (DHB),

GC/MS Second GalA transferase required for

core LPS biosynthesis (Fresno, et al.,

2007)

Klebsiellapneumoniae Lipid A R-TOF

(CHCA)

Study of secondary acylation contribution to sensitivity to

antibacterial peptides

(Clements, et al., 2007)

Lactobacillusdelbrueckii ssp.

bulgaricus LBB.B26EPS TOF (DHB),

GC/MS, NMR

Structural determination from yoghurt. Branched pentasaccharide

repeat.

(Sánchez-Medina, et al.,

2007a) Lactobacillus

delbrueckii ssp.bulgaricus LBB.B332

EPS TOF (DHB), GC/MS, NMR

Structural determination from yoghurt. Linear pentasaccharide

(Sánchez-Medina, et al.,

2007b)

Moraxella catarrhalis LOS TOF (DHB),

GC/MS, NMR Mutant lacking O-chain shows linker

essential for membrane integrity (Peng, et al.,

2007)

Moraxella catarrhalis LOS

TOF (DHB) Glycans (per-Me), GC/MS,

NMR

Investigation of the functional role in biosynthetic glycosyltransferases

(Peak, et al., 2007)

Moraxella catarrhalis 26404 LPS TOF (1,2-

DHB)

Galactose residues shown to form the epitope recognized by the

bactericidal antiserum from conjugate vaccination

(Yu, et al., 2008b)

Neisseriagonorrhoeae LOS TOF (DHB)

Structural requirements for monoclonal antibody 2-1-L8

recognition

(O'Connor, et al., 2008)




Neisseriameningitidis NMA

Z2491(L9 immunotype)

LPS TOF/TOF

(DHB), GC/MS, NMR

Structural determination. Identical to L4 LOS but without sialic acid.

(Choudhury, et al., 2008b)

Neisseriameningitidis LPS TOF/TOF

(THAP)

Modification of LOS with phosphoethanolamine by LptA

enhances meningococcal adhesion to human endothelial and epithelial

cells

(Takahashi, et al., 2008a)

Neisseriameningitidis strains LOS TOF

Characterization of LOS to immuno-purify IgG that binds lacto-N-

neotetraose

(Estabrook, et al., 2007)

Plesiomonas shigelloides Strain 302–73 (Serotype

O1)

O-chain TOF (DHB), GC/MS, NMR Structural determination (Pieretti, et al.,

2008)

Porphyromonas gingivalis Lipid A TOF, GLC Study of the distribution of acyl

chains (Bainbridge, et

al., 2008)

Porphyromonas gingivalis W50

LPS, Lipid A

TOF (nor-harmane),

GC/MS, NMR

Structural identification of second LPS with phosphorylated mannan

(Rangarajan, et al., 2008)

Propionibacteriumfreudenreichii 109, P. freudenreichii

111, P.m thoenii 126

EPS R-TOF

(DHB), GLC, GC-MS, NMR

Structural determination. All three bacteria have the same carbohydrate

chain.

(Dobruchowska, et al., 2008)

Proteus vulgaris TG276-1 O-chain

TOF (3,5-DHB), GLC,

NMR

Unique structure. New serogroup (O53) proposed

(Arbatsky, et al., 2007)

Pseudomonas aeruginosa and

Bordetella pertussis

Mono-saccharide from LPS

TOF (DHB) Biosynthesis of an Ac-sugar in LPS of both species occur via identical

scheme despite different genes

(Westman, et al., 2008)

Pseudomonas aeruginosa, PA103,

wbjE mutant

LPS (Low and high

MW)

TOF (sDHB), GC/MS, NMR

Structural determination and biosynthesis

(Choudhury, et al., 2008a)

Pseudomonas aeruginosa Lipid A TOF (CMBT)

Unique lipid A modifications (AraN, palmitate) isolated from the airways

of patients with cystic fibrosis

(Ernst, et al., 2007)

Pseudomonas aeruginosa LPS TOF (CHCA)

Natriuretic peptides specifically modify lipopolysaccharide

biosynthesis

(Veron, et al., 2007)

Pseudoalteromonasnigrifaciens Lipid A

TOF (DHB, CHCA),

NMR, TLC Structural determination (Volk, et al.,

2007)

Pseudomonas tolaasii, P. reactans

and Burkholderia gladioli pv. agaricicola

Lipid A TOF Perspective - structural identification of various metabolites

(Andolfi, et al., 2008)

Pseudomonas Sp. OX1 O-chain L-, R-TOF

(DHB), NMR

Structural determination. Bacterium reduces azo dyes but modifications of monosaccharide composition and

architecture occur to O-chain.

(Leone, et al., 2008)

Rhizobium etli CE3 LPS R-TOF

(THAP), GC/MS

LPS from Phaseolis vulgaris root nodules differs from that from lab

cultures

(D'Haeze, et al., 2007)

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b. Glycan analysis following removal of the ceramide. Enzy-matic removal of the ceramide (3/20) from glycosphingolipidsremoves the heterogenicity contributed by the lipid portion andallows the glycan to be analyzed by conventional techniques.A method for examination of the glycan portion fromglycosphingolipids, developed by Parry et al. (2007a), usedceramide glycanase to cleave the glycans, which were thenexamined as their permethyl derivatives by MALDI-TOF and

MALDI-TOF-TOFMS fromDHBor CHCA. Themethod can becombined with established techniques for the analysis of N- andO-linked glycans for examination of carbohydrates in the samesample and has been applied to the identification of these threeglycan types in mouse brain. Endoglycoceramidase was used byMeyer et al. (2007) to release glycans from Schistosomamansoniglycosphingolipids for analysis byMALDI-TOFand to show thatthey were recognized by the C-type lectin, L-SIGN. A novel


Rhizobiumleguminosarum

3841O-chain

TOF (DHB), ESI-Q-TOF,

GC/MS, NMR

Structural determination. Ident. of several unusual sugars, including 6-deoxy-3-O-Me-D-talose, 2-NAc-2- deoxy-L-quinovosamine and a new

sugar, 3-acetimidoylamino-3-deoxy-D-gluco-hexuronic acid

(Forsberg & Carlson, 2008)

Rhizobium rubi Outer core TOF (THAP), GC/MS, NMR

Structural determination. Lewis B epitope

(Gargiulo, et al., 2008)

Rhizobium sp. EPS R-TOF (DHB)

Production and composition of EPS synthesized by a Rhizobium isolate from Vigna mungo (L.) Hepper root

nodules

(Mandal, et al., 2007)

Salmonella Dakar O-chain TOF (DHB),

GLC, GC/MS, NMR

Structural determination and Smith degradation

(Kumirska, et al., 2008)

Salmonella Dakar (serogroup O:28) O-chain

TOF (DHB), GLC, GC/MS,

NMR

Structural determination. Branched pentasaccharide repeat.

(Kumirska, et al., 2007)

Salmonella enterica Serovar

TyphimuriumLipid A TOF (CMBT,

DHB)

Bacterium modifies lipid A in response to environment by adding

AraN and/or phosphoethanolamine to phosphate groups.

(Manabe & Kawasaki, 2008)

Salmonella enterica Lipid A TOF (CMBT, DHB)

Effect of modifications to lipid A on activity of 3-O-deacylase

(Kawasaki, et al., 2007a)

Salmonella strainATCC 14028 msbB Lipid A TOF, GLC

EGTA and polymyxin resistance on msbB Salmonella conferred by

decorating lipid A with phosphoethanolamine

(Murray, et al., 2007)

Shigella flexneri M90T serotype 5 and galU mutant

R-LPSTOF (DHB,

THAP),GC/MS, NMR

Structural determination in connection with vaccine

development. Mutant used to assign glycine residue to O-6 of the second

heptose of inner core

(Molinaro, et al., 2008)

Vibrio fischeri Lipid A R-TOF

(CMBT), GC/MS

Characterization of htrB and msbBmutants

(Adin, et al., 2008)

Vibrioparahaemolyticus

O3:K6LOS TOF (DHB),

ESI, NMR

Identification of novel sugar - 5,7-Di-NAc-8-NH2-3,5,7,8,9-pentadeoxy-D-

glycero-D-galacto-non-2-ulosonic acid in new pandemic strain

(Mazumder, et al., 2008)

Xanthomonas Campestris LPS

L-, R-TOF (THAP (intact

LPS)), DHB carbohydrates,

GLC, GC/MS, NMR

Acylation and phosphorylation pattern of lipid A shown to strongly influence ability to trigger the innate

immune response in Arabidopsis

(Silipo, et al., 2008)




endoglycoceramidase from Rhodococcus equi that hydrolyzedthe glycan ceramide bond of 6-gala- but not ganglio-, globo-, orlacto-series glycosphingolipids has been identified (Ishibashiet al., 2007b). MALDI-TOF analysis was used to verify theglycan released from tri-galactosylceramide. Details of a glycanrelease method using endoglucoceramidase (EC3.2.1.123) havebeen published (Ito, 2008).

c. Combined TLC-MALDI-TOF. TLC is extensively used forseparation of glycosphingolipids. A method for interfacing TLCwith IR-MALDI has been described by Distler et al. (2008a).GSLs were separated by TLC and stained with orcinol or anti-GSL antibodies in parallel runs. For mass spectrometry, theimmuno-stained bands were soaked in ammonium acetate(pH 3.6) for 2 hr and dried. The TLC fixative was removed withchloroform and the plates were cut into 15mm� 40mm sectionsand stuck to the MALDI probe with double-sided tape. Glycerol(matrix) was applied to the immuno-positive bands and IR-MALDI spectra were acquired with a Er-YAG laser. Detectionlimits were reported to be below 1 ng and the systemworkedwithcrude lipid extracts. The method was applied to various systemsincluding GSLs from human pancreatic and hepatocellularcarcinomas. In a method using more conventional UV-MALDI,stem cell lipids have been separated by high-performance TLC,sprayed with DHB and imaged by MALDI. Gangliosides couldbe seen in both positive and negative ion modes. Sensitivity wassaid to be much higher than could be obtained by conventionalstaining protocols (Fuchs et al., 2008b). A similar study withlipids from porcine brain has also been reported (Fuchs et al.,2008a). Goto-Inoue et al. (2008) have blotted the glycosphingo-lipid GM1 (66) from a TLC plate onto a PVDF membraneand imaged it at a sensitivity of 1 pmol with DHB as the matrix.Use of a QIT mass spectrometer allowed MSn spectra to becollected for structural analysis. Collisional cooling was used tostabilize the sialic acids and the method was used to analyzeGSLs derived from the brain of a human patient with Tay-Sachsdisease.

d. Other methods. A method for the quantitative analysis ofserum sulfatides has been described (Li et al., 2007a). Thecompounds were extracted with hexane/iso-propanol dried andthe acyl groups were removed with NaOH in 90% methanol(1508C, 30min). The resulting lyso-sulfatides were extractedwith a C18MicroTip and analyzed byMALDI-TOF fromCHCAallowing quantitation of serum sulfatides from one hundred- to50-mL serum specimens within 1 working day. A quantitativemethod has been described for ceramide monohexoside and

sphingomyelin using sphingosylphosphorylcholine as the inter-nal standard (Fujiwaki, Tasaka,&Yamaguchi, 2008) andDHB asthe MALDI matrix. The calibration curve for ceramidemonohexoside was linear over the range 5–150 ng and themethod was used to measure accumulations of the glycolipids inliver and spleen samples from patients with Gaucher disease.

Landoni et al. (2008) have coupled the fluorescent tag,N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)aminocaproic acid, (67, NBD)via a C6 linker to the amino group of sphingoid bases such assphingosine (1/75, NBD-ceramide), glucosyl-sphingosine andgalactosyl-sphingosine and found that these compounds producegood signals without a matrix. Furthermore, when added toglycosphingolipid samples, NBD-ceramide produced spectra ofother glycolipids in the mixture, such as lactosyl-, gangliote-traosyl-, and globotetraosyl-ceramide. The method was appliedto the identification of glycosphingolipids from the epimastigoteforms of Trypanosoma cruzi. Kholodenko et al. (2008) have usedGM1 containing a diazocyclopentadien-2-ylcarbonyl group (68)attached to the sialic acid in a photoaffinity labeling study toinvestigate the binding sites in interleukin-4. The resultingcomplex was digested with Glu-C and the fragments analyzed byMALDI-TOF. Further examples of the analysis of glycosphin-golipids are listed in Table 13.

3. Other glycolipids

Many other types of bacterial glycolipids have been investigated;these are listed in Table 14. Extraction and clean-upmethods vary

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TABLE 13. Use of MALDI MS for examination of glycosphingolipids

Species Organ Mathods1 Notes Reference

Drosophilamelanogaster Whole flies TOF/TOF (DHB -ve,

ATT +ve)

Study of contributions of GalNAc-transferases A and B to extensions

of Cer-Glc-Man-GlcNAc

(Stolz, et al., 2008)

Fungi (various)

Sousei river sludge (water

treatment) R-TOF (DHB), GC/MS Structural determination. As

possible signatures of fungi (Tsuge, et al.,

2008)

Halocynthia roretzi Total

R-TOF (DHB, coumarin 120), NMR,

TLC, GC/MS

Structural determination, Novel GalpA moiety attached to ceramide

(Ito, et al., 2007b)

Human Erythrocytes QIT-TOF (DHB), Per-Me, GC/MS

Structural determination of unique globoside elongation product in

NOR phenotype

(Duk, et al., 2007)

Mouse Embryonic fibroblasts R-TOF/TOF (CHCA)

GM3 Knock-out mice have activated alternate pathway

producing GM1b, GM1b, GD1α

(Shevchuk, et al., 2007)

Human Colon-

carcinoma cell line, Colo205

R-TOF/TOF (DHB), Q-TOF (CHCA), QIT

(DHB), Glycans (per-Me)

Shows that type 1 chains of Lac-Cer can be extended in cancer. Glycans by ceramide digestion

(Fan, et al., 2008)

Human Pancreatic cancer

IR-MALDI-TOF (glycerol)

Elevated CD75s and iso-CD75s gangliosides in pancreatic cancer

(Distler, et al., 2008b)

Human Peripheral

blood neutrophils

QIT-TOF, TOF (ATT), LC-MS/MS

To determine receptors for E-selectin

(Nimrichter, et al., 2008)

Mouse Colon

carcinoma MC-38 cells

TOF/TOF (ATT) P-selectin shown to mediate

metastatic progression through binding to sulfatides on tumor cells

(Garcia, et al., 2007)

Mouse Embryonic stem cells

MALDI (DHB), glycans (2-AB)

Analysis of glycans released with ceramide glycanase. Lc3-synthase gene involved in lacto-series GSLs - essential for cellular development

(Biellmann, et al., 2008)

Mouse Tissue MALDI-ion trap Description of method for interfacing TLC

(Miyazaki, et al., 2008)

Mouse Brain TOF Mutant lacking 2-OH fatty acyl groups showed normal myelin

formation but rapid degeneration

(Zöller, et al., 2008)

Plasmodiumfalciparum

Whole organism TOF (-ve nor-harmane) Identification of

sulfoglycosphingolipids (Landoni, et

al., 2007)

Rat Brain TOF Ceramide, ceramide-monohexoside

and sphingomyelin elevated in model of epilepsy

(Ma, et al., 2007)

Schistosoma mansoni Eggs R-TOF (ATT)

The C-type lectin L-SIGN differentially recognizes glycan

antigens on egg glycosphingolipids and soluble egg glycoproteins

(Meyer, et al., 2007)

General - QIT-TOF Brief experimental details of

endoglucoceramidase release and 2-AP derivatization

(Suzuki, 2008)

1Format (not all items present): MALDI method (matrix), compounds studied (derivative) other methods.



TABLE 14. Use of MALDI MS for the analysis of bacterial glycolipids

Species Glycolipid Methods1 Notes Reference Corynebacterium

glutamicumpimB0 mutant

Lipomannan TOF, TOF/TOF, MS/MS

Structural characterization Functional properties of a novel lipomannan variant

(Mishra, et al., 2008)

Corynebacterium glutamicum ubiA

mutant

Partiallyarabinosylated

lipoarabinomannan

TOF (DHB), Native (per-Me)

Structural determination. Postulation of alternative

pathways for Arafdelivery

(Tatituri, et al., 2007)

Mycobacteriumabscessus and M.

chelonae Glycopeptidolipids TOF Genomics of biosynthesis (Ripoll, et al.,

2007)

Mycobacteriumavium

Glucose monomycolate R-TOF (DHB)

Trehalose dimycolate changes to Glc-

monomycolate in the presence of glucose

(Matsunaga, et al., 2008)

Mycobacteriumavium Glycopeptidolipids R-TOF (DHB),

GC/MS Genetics for biosynthesis

of serovar 8 (Miyamoto, et

al., 2008)

Mycobacteriumbovis

Lipoarabinomannan/ lipomannan R-TOF (DHB)

Targeted glycolipids induce CD4 T-cell

differentiation (Ito, et al., 2008)

Mycobacteriumbovis (Calmette

Guerin)

Phosphatidyl-myo-inositol mannosides TOF Structural determination

(Hsu, et al., 2007a, Hsu, et

al., 2007b)

Mycobacteriumintracellulare Glycopeptidolipids TOF, TOF/TOF

(DHB)

Characterization of two novel methyl-transferase genes that determine the serotype 12- structure

(Nakata, et al., 2008)

Mycobacteriumintracellulare Glycopeptidolipids TOF, TOF/TOF

(DHB) Structural determination

of serotype 16 GPL (Fujiwara, et al.,

2008)

Mycobacteriumsmegmatis Glycopeptidolipids TOF/TOF

(DHB)

Strain deficient in selective incorporation of

hydroxylated glyco-peptidolipids in cell wall

forms mutant biofilm.

(Mukherjee & Chatterji, 2008)

Mycobacteriumsmegmatis Crude lipid fraction TOF Study of natural variation

on phenotype (Kocíncová, et

al., 2008)

Mycobacteriumspp. Lipoarabinomannan

R-TOF/TOF(DHB), glycans (per-Ac and Me)

Mannose capping by multifunctional

mannosyltransferase

(Kaur, et al., 2008)

Mycobacteriumtuberculosis Lipoarabinomannan TOF (DHB,

NMR

Isolates with truncated lipoarabinomannan. Altered phagocytosis

(Torrelles, et al., 2008)


Phosphatidylinositol mannosides

TOF (CHCA) GC/MS

Identification of new mannosyl transferase

(Lea-Smith, et al., 2008)


Phthiocerol and phthiodiolone

dimycocerosates TOF (DHB)

Identification of ketoreductase involved in

biosynthesis

(Simeone, et al., 2007)

Streptococcus pneumoniae Lipoteichoic acid TOF (DHB),

ESI, MS/MS New structure determined (Seo, et al., 2008)

1Format (not all items present): MALDI method (matrix), compounds run (derivative), other methods.

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but, as an example, lipoarabinomannan has been recovered byestablished methods from dried supernatant obtained fromCorynebacterium glutamicum (Post & Gibson, 2007). Thismethod involved delipidation, cellular disruption and filtrationafter which, the dried supernatant was treated with a hot 80%(w/w) phenol/H2O biphasic wash at 708C for 1 h, followedby several protease treatments. The lipoglycan fraction wasrecovered following extensive dialysis against deionized water,purified and the glycans were released with TFA. MALDI-TOFanalysis showed a broad peak in the region of 15 kDa.

Possibly the most complex glycolipid for which MALDI-TOFMS has helped to investigate is themycolyl arabinogalactan(69) from Mycobacterium tuberculosis. A spectrum of theintact molecule was not obtained but MALDI-TOF wasused to investicate various carbohydrate fractions after depoly-merization.

I. Glycosides and Other Natural Products

Although MALDI-TOF has been used on several occasions forthe structural analysis of glycosides, the majority of reportedwork used electrospray. Several instances of fast atom bombard-ment (FAB) have also been reported. Compounds examinedduring the review period are listed in Table 15.

MALDI-TOF MS and other mass spectral techniques havebeen used to detect terpene glycosides at low concentrationfor characterization of grape varieties and wine, overcominglimitations of previous techniques for the detection of character-istic molecules that are only present in trace amounts (Nasi et al.,2008). A method for comparing wines by monitoring anthocya-nins and other constituents has been described (Carpentieri,Marino, & Amoresano, 2007). DHB proved to be the best matrixand profiles of 80 constituents, many of them glycosylatedcompoundswere reported for 20wines. Twenty four furanostanol(diosgenin, 70) saponins with glucose, xylose, and rhamnosehave been identified from the desert date (Balanites aegyptiaca)and the authors commented that ‘‘The results suggest thatMALDI-TOF/MSwith positive ionmode is particularly effectivefor determining the metabolites of saponins in B. aegyptiacaplant tissues. MALDI-TOF/MS not only verified the results ofthe LC (RI)-ESI/MS, but also identified additional saponins thatare now systematically organized in a database.’’

Several successful syntheses of glycosides have been carriedout with products analyzed byMALDI-TOFMS. These are listedin Table 16.

XIV. MEDICAL APPLICATIONS

There is increasing use of MALDI MS in research into variousaspects of disease, particularly the detection of cancer bio-markers. Thus, Kam and Poon (2008) have reviewed thepotentials of glycomics in biomarker discovery and glycanmapping is also included in a review of metabolic profiling, thatcovers topics such as the use ofMALDI-TOFMS for the study ofglycosylation in prostate cancer (Novotny, Soini, & Mechref,2008). A detailed review of the constituents of the human serumN-glycome and changes to theN-glycans in various disease stateshas been published (Klein, 2008) and Haslam et al. (2008) havereviewed the current status of glycomics of the mammalianimmune system including strategies and methods for character-ization of N- and O-glycans.

A. Biomarkers

Changes in both N- and O-linked glycans such as increasedfucosylation have been linked with several disease states,



TABLE 15. Use of MALDI MS for examination of natural compounds

Species Compound Methods1 Notes Reference

Aesculus turbinata Triterpene saponins (isoescins)

TOF, NMR, HPLC

Structural determination, three new, from seeds

(Yang, et al., 2008b)

Alvaradoahaitiensis

Anthracenone C-glycosides

(Alvaradoin)

TOF/TOF (DHB), FAB, NMR, HPLC

Structure determination and toxicity

(Phifer, et al., 2007)

Arabidopsisthaliana

Sinapoylated anthocyanins and

biosynthetic intermediates

R-TOF(CHCA) Genetics of biosynthesis (Fraser, et al.,

2007)

Argania spinosa Triterpenoid

saponins, (arganine L , O , P , Q and R)

TOF (DHB), ESI, ORD

Structural identification of five new saponins,

(El Fakhar, et al., 2007)

Asparagusofficinalis Anthocyanins

R-TOF(THAP), FT-ICR, TLC,

NMR

Structural determination. Two cyaniding glycosides identified

(Sakaguchi, et al., 2008)

Aspergillusfumigatus

Glycosylinositol phosphoryl-ceramides

TOF Structural determination (Toledo, et al., 2007)

Astragalus campylosema

Boiss ssp.campylosema

Cycloartaneglycosides

TOF (CHCA), ESI, NMR

Structural identification, four new, three known

(Çalis, et al., 2008)

Astragalus eremophilus

Cycloartaneglycosides TOF, NMR

Structural determination, Eleven new. Some caused cell death by

apoptosis

(Perrone, et al., 2008)

Bacopa monnieri Triterpenoid saponins

TOF/TOF (CHCA), QIT-TOF (THAP), AP-MALDI

(CHCA) trap, LC-ESIMS

Use of different MS techniques for structural determination

(Zehl, et al., 2007)

Balanitesaegyptiaca

(desert date)

Furanostanol(diosgenin)

saponins

TOF (DHB), ESI

Structural determination. 24 compounds

(Chapagain & Wiesman, 2008)

Beverage from fermented plant

extract

Di- and trisaccharides TOF, NMR Structural determination. 14

compounds (Okada, et al.,

2008a)

Beverage from fermented plant

extract

O-β -D-Fructopyranosyl-

(1→2)-D-glucopyranose

TOF, NMR Structural determination and properties (sweetness etc.)

(Okada, et al., 2008b)

Bombyx mori L.(Male silkworm)

pupae

Dimethyladenosine-5’-L-arabinose

TOF, EI, GC/MS, NMR

Structural determination of compound for potential

treatment of erectile dysfunction

(Ahn, et al., 2008)

Camellia sinensis (flower buds)

Triterpenoid glycosides

MALDI, FAB, HPLC, TLC,

NMR

Structural determination. Four compounds

(Yoshikawa, et al., 2008a)

Cephalaria gigantea

Oleanane-type saponins,

(giganteosides L, M and N)

TOF, ESI, NMR

Structural determination from roots

(Tabatadze, et al., 2007)

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Chlamydomonasreinhardtii (green alga), Cyclotellameneghiniana

Mono- and digalactosyl

diacylglycerol

TOF (DHB), TLC

Lipid and glycolipid composition determined by

MALDI-TOF and TLC

(Vieler, et al., 2007)

Ciona intestinalis (ascidian) Glycosphingolipid TOF, NMR Structural determination (Yamada, et al.,

2007b)

Conyza blinii Labdane

diterpenoid arabinoside

FTMS Structural determination (Su, et al., 2008)

Crassocephalummannii

Labdane diterpene glycosides

TOF, NMR, HPLC

Structural determination. Two compds. Anti-cyclooxygenases

(Hegazy, et al., 2008)

Cryptococcus neoformans

Glycoinositol-phosphoryl ceramide

TOF (-ve, nor-harmane), ESI Structural determination (Gutierrez, et al.,

2007)

Cucumariaokhotensis (sea

cucumber)

Triterpene glycosides

TOF (DHB), HPLC, NMR

Structural determination, three new. Seven known compounds

(Silchenko, et al., 2008)

Cyperus rotundusL. Steroid glycoside

TOF, EI, HPLC, NMR,

UV, TLC

Structural determination. Cytotoxic properties

(Sayed, et al., 2007)

Distolasterias nipon (far Eastern

starfish)Steroid glycosides TOF, NMR

Structural determination, two new, induced neuroblast differentiation in mouse cell culture.

(Kicha, et al., 2008b)

Durinskia sp. Long-chain polyol TOF, FAB MS/MS, ESI Structural determination (Kita, et al.,

2007a)

Elsholtziarugulosa

Maltol and cyanogenicglycosides

TOF, FAB, NMR

Structural identification of two new and 11 previously known

compounds (Li, et al., 2008c)

Erylus formosus Steroid glycosides (erylosides)

TOF (CHCA, HPLC, ORD,

NMR

Identification of nine new compounds

(Antonov, et al., 2007)

Erylus goffrilleri Triterpene glycosides

(erylosides)

TOF (CHCA, HPLC, ORD,

NMR

Identification of eight new compounds

(Afiyatullov, et al., 2007)

Eryngiumyuccifolium

Triterpenoid saponins

(eryngiosides A-L)

TOF, ESI, NMR

Structural determination. 12 eryngiosides plus seven other compounds; four were known.

(Zhang, et al., 2008i)

Evasterias retifera(Pacific starfish)

Steroid glycosides. (evasteriosides A

and B)

TOF (CHCA), NMR

Structural determination of two new compounds.

(Levina, et al., 2008)

Flax seed hulls Flavonoid TOF/TOF (DHB)

Structural determination of herbacetin diglucoside

(Struijs, et al., 2007)

Globularia alypum

Phenylethyl glycosides

R-TOF, NMR, IR, ORD

Structural determination of three new glycosides

(Kirmizibekmez, et al., 2008)

Gypsophila oldhamiana

Triterpenoid saponins,

(gypsosaponins A-C)

TOF, FAB, NMR, HPLC

Structural determination from roots

(Zheng, et al., 2007)

Hedysarum theinum (roots)

Isoflavonoids and similar compounds

TOF (DHB), IR, OR, NMR Structural determination (Nechepurenko,

et al., 2008) Helleboruscaucasicus

(evergreen plant) Steroid glycosides

TOF (CHCA), ESI, ORD, GC, HPLC

Structural determination four new compounds

(Bassarello, et al., 2008)

Hippasteria kurilensis (Far

Eastern starfish)

Steroidal glycosides

TOF (CHCA), NMR, HPLC

Structural determination. First two-sugar chains from starfish.

(Kicha, et al., 2008a)




Hypsizygus marmoreus

(edible mushroom)

Glycosylinositol-phosphoceramides

TOF (7-NH2-4-Me-

coumarin), GC/MS, NMR

Structural determination (Itonori, et al., 2008)

Ipomoea tyrianthina Tyrianthinic acids

TOF (CHCA), FAB, NMR,

HPLC

Structural determination, four antimycobacterial compds.

(León-Rivera, et al., 2008)

Klebsiellapneumonaie

RYC492Protein toxin TOF Genetics of biosynthesis (Nolan, et al.,

2007)

Lethasterias fusca (Far Easterm

starfish)Steroid glycosides TOF, NMR Structural determination (Ivanchina, et al.,

2008)

Linckia laevigata(Viet Namese

starfish)

Sulfated steroid glycoside

TOF (DHB), ESI, NMR Structural determination (Kicha, et al.,

2007)

Lycopersiconesculentum cv.

Cedrico (tomato)

Triterpenoid glycosides

R-TOF(CHCA),

NMR, HPLC

Structural determination and surfactant properties.

(Yamanaka, et al., 2008)

Mycobacteriumleprae

Trehalose 6,6’-dimycolate R-TOF (DHB) First identification in this

bacterium (Kai, et al.,

2007)

Oceanapia sp.

Two-headed sphingolipid-like

compounds (rhizochalin B and rhizochalinin B)

TOF (DHB), ESI, NMR Structural determination (Makarieva, et

al., 2008)

Oryza sativa L. japonica (rice) Anthocyanins

TOF (MALDI “electrosprayionization”)

Structure determination and aldose reductase inhibitory

activity

(Yawadio, et al., 2007)

Paris polyphyllavar. yunnanensis

Phenylpropanoid glycosides

(parispolysides F, G) MALDI Structural determination (Wang, et al.,

2007c)

Paris polyphyllavar. yunnanensis

Phenylpropanoid glycoside

MALDI, NMR, IR

Structural determination. Cytotoxic against mouse lung

adenocarcinoma cell line (LA795).

(Yan, et al., 2008)

Paulownia coreana Uyeki

(inner bark)

Phenylethanoid glycosides

R-TOF, NMR, LH-20, TLC

Structural determination. Two known, one new structure.

(Kim, et al., 2007c)

Paulownia coreana (leaves)

Phenylpropanoid glycosides

TOF, FAB, TLC, NMR Structural determination (Kim, et al.,

2008c) Paulownia

tomentosa var.tomentosa (wood)

Phenylethanoid glycosides

R-TOF, NMR, HPLC, TLC

Structural determination. Three known, one new structure. (Si, et al., 2008)

Phaeodactylumtricornutum

(Marine diatom)

Galactosyldiacylglycerols

TOF-TOF, ESI, NMR,

HPLC

Structural determination, Two apoptosis-inducing compds.

(Andrianasolo, et al., 2008)

Polygonummultiflorum

(roots)

Stilbene glucoside gallate

TOF, ESI, FAB, NMR,

OR

Structural determination. Weak acetylcholineesterase inhibitor

(Kim, et al., 2008a)

Porphyridium purpureum (Microalga)

Sulfoquinovosyl-diacyl glycerol

QIT-TOF (DHB, CHCA)

Structural determination. First investigation of purified

compound

(Naumann, et al., 2007)

Pseudozymaantarctica

Mono-acylated mannosylerythritol

lipid

TOF (CHCA), GC/MS, NMR

Structural characterization and surface-active properties

(Fukuoka, et al., 2007c)

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particularly those involving inflammation. Several suitabletechniques have been reported during the review period. Ruhaaket al. (2008) have used a 96-well plates format to remove N-glycans from 10mL of plasma glycoproteins with PNGase F andto label them directly with 2-AA. The labeled glycans wereseparated by HILIC chromatography for analysis by MALDI-

TOF MS, HPLC with fluorescence detection or capillaryelectrophoresis-MS. Forty-seven glycans could be measuredconsistently by MALDI-TOF and CE-ESI-Q-TOF MS. Themethodwas claimed to be faster than that reported by Royle et al.(2008) that mainly relied on HPLC for glycan profiling. In themethod reported by Ruhaak et al. the 2-AA labeling procedure


Pseudozymaantarctic

Pseudozymarugulosa

Tri-acylated mannosylerythritol

lipid


Structural characterization and surface-active properties

(Fukuoka, et al., 2007a)

Pseudozymacrassa (Yeast)

Mannosylerythritol lipids

TOF (CHCA), NMR

Structural determination. Biosurfactants

(Fukuoka, et al., 2008b)

Pseudozymashanxiensis

Di-acylated mannosylerythritol

lipid


Structural characterization, produced from soybean oil

(Fukuoka, et al., 2007b)

Pseudozymatsukubaensis

(Yeast)

Mannosylerythritol lipid-B


Structural determination. Config. different to known

(Fukuoka, et al., 2008a)

Rhizochalinaincrusyata

(Marine sponge)

Two-headed glycosphingolipid

TOF, FAB, ESI, NMR,

CD

Structural determination and revision of structure

(Makarieva, et al., 2007)

Rubussp.(Blackberry) Ellagitannins TOF (DHB)

Structural determination - identification of several new

compounds

(Hager, et al., 2008)

Rubus suavissimus S. Lee (Chinese

sweet tea)Ellagitannins TOF, TLC,

NMR, UV, IR Structural determination. -

Amylase inhibition (Li, et al., 2007b)

Scrophularia crypthophila Resin glycosides FTMS, ESI,

NMR

First report of this type of glycoside from

Scrophulariaceae

(Çalis, et al., 2007)

Stevia rebaudiana Terpenoid glycosides

TOF (DHB), NMR

Low calorie sugar substitute. From plants grown in Russia and

Ukraine (Crimea)

(Kovylyaeva, et al., 2007)

Streptomyces flavoviridis ATCC

21892

Zorbamycin (antitumor properties)

FTMS, ESI, NMR

Structure determination and yield improvement

(Wang, et al., 2007b)

Synapta maculate (Vietnamese sea

cucumber)

Triterpene (holostane) glycosides

TOF (CHCA), NMR

Structural determination, two new. Moderate cytotoxic

activity, against HeLa cells

(Avilov, et al., 2008)

Triumfettacordifolia A. Rich

(shrub)

Ceramides (from stems)

TOF, FTMS (DHB)

Structural determination. Named Triumfettamide and

Triumfettoside Ic

(Sandjo, et al., 2008)

Trofodiscus über (Far east starfish) Steroid glycosides TOF (CHCA),

HPLC, NMR Structural determination (Levina, et al., 2007)

Vaccinium axillare Flavonoids TOF Structural determination (Mechikova, et

al., 2008) Xanthoceras

sorbifolia Bunge (husks)

Triterpene saponins TOF, ESI, GC/MS, NMR

Structural determination. Four new. Anti-ovarian cancer

(Chan, et al., 2008)

Xanthocerassorbifolia Bunge

Steroid saponin (anti-tumor properties)

R-TOF, HPLC

Removal of diangeloyl (CO-C(Me)=CH-CH3 groups from C21,22 positions abolishes

biological activity

(Chan, 2007)




could be applied directly to the released glycan solution whereastheRoylemethodused 2-AB labeling that required the samples tobe dried before the reductive amination labeling procedure. AMALDI-TOF-based method for identifying the glycoproteinsfrom liver carrying increased fucosylation has been reported byDai et al. (2007) who first fractionated the glycoproteins withLens culinaris agglutinin, a lectin that binds specifically to thisstructural feature.

Detection of possible cancer markers is a topic pursuedby several laboratories. Kang et al. (2007) have reported aquantitative method for measuring differences in abundance ofN-glycans obtained from breast cancer patients and healthycontrols. The glycoproteins were reduced and alkylated prior totreatment with trypsin and PNGase F. The released glycans werecleaned with C18 and activated carbon and either permethylated(control samples) or per-[2H3]-methylated (breast cancer sam-ples) using methyl iodide and the author’s NaOH bead column.Both N- and O-glycans were also obtained from a breast cancercell line. To eliminate potential problems associated withquantitative differences in permethylation and per-[2H3]-meth-ylation, tests were carried out with ribonuclease B over a range ofconcentrations. No significant differences were found with the

intensity ratios between 0.125 and 6 with reproducibility betterthan 30%RSD. The results from the breast cancer study revealedan increase in N-glycosylation in the cancer patients. Of 60O-linked glycans, 32 showed a decrease in abundance whereasthe remainder were detected in higher abundance.

Ueda et al. (2007) have developed a method for biomarkeridentification in serum involving first immunodepletion of sixhigh-abundance proteins followed by targeted enrichment ofglycoproteins by lectin column chromatography and quantitativeproteome analysis using 12C6- or

13C6-NBS (2-nitrobenzenesul-fenyl, 71) stable isotope labeling. Detection and measurement ofthe labeled glycoproteins was made by MALDI-QIT-TOF massspectrometry. N-glycans were then released from haptoglobin,one of the target glycoproteins, and identified by MALDI-QIT-TOF. Significant differences in core 1! 6-fucosylation have alsobeen found between samples from lung cancer patients andcontrols. Zhao et al. (2007a), in a study using both MALDI andLC-ESI-MS/MS, detected 44 N-glycans in human serum thatappeared to be associatedwith pancreatic cancer. They containedincreased branching, fucosylation and sialylation compared tocorresponding samples from controls. Barrabes et al. (2007)using MALDI profiling, negative ion ESI-MS/MS and ultra

TABLE 16. Use of MALDI MS to monitor the products of glycoside syntheses

Source Glycans Methods1 Notes Reference

Anemone flaccida“Di Wu”,

Triterpenoid glycoside

(Flaccidoside II)

TOF(CHCA)

Synthesised from protected-D-Xylp, α-L-Rhap β-D-Glcp and

oleanolic acid

(Cheng, et al., 2008b)

Beta vulgaris and Achyranthes fauriei


(Betavulgaroside III)

MALDI Synthesis in 31 steps from L-

arabinose, D-glucose and oleanolic acid

(Zhu, et al., 2008b)

Bolbostemmapaniculatuma and

Actinostemmalobatum


(Lobatoside E) MALDI

Potent antitumor cyclic triterpene saponin. Removal of glutarate bridge kills activity.

Synthesised from oleanolic acid

(Zhu, et al., 2008a)

Brassica oleracea var. capitata

1-Methoxy-1-(α-D-ribofuranosyl)- and

1-(β-D-ribofuranosyl)-

brassenin B

TOF Synthesis from 1-substituted

indole-3-carboxaldehydes. Anti-cancer

(Čurillová, et al., 2008)

Dioscorea villosa,(Wild Yam)

Steroidal glycoside (Dioscin) MALDI

Use of glycosyl ortho-alkynylbenzoates as donors

under the catalysis of Ph3PAuOTf

(Li, et al., 2008g)

Hydrangeamacrophylla

3-O-(2-O-β -D-xylopyranosyl)-β -

D-gluco-pyranosylcyanidin

TOF To clarify the chemical

mechanism of the color change in flowers

(Yoshida, et al., 2008)

Quillaja saponariaTriterpenoid

glycoside (QS-7-Api)

MALDI

Constituent of the bark of Quillaja saponaria. Used as

adjuvant in vaccines with no side effects.

(Deng, et al., 2008)

Various 6’-O-Substituted dioscin derivatives TOF

Synthesis and study of cytotoxicity and haemolytic

activity (Li, et al., 2007e)

1Format (not all items present): MALDI method (matrix). ‘‘MALDI’’ used when instrument not specified.

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performance liquid chromatography (UPLC) have shown anincrease in core fucosylation of N-glycans in ribonuclease1 associated with this disease. Increased fucosylation has alsobeen found to be associated with prostate cancer (Kyselovaet al., 2007). In this study, glycans were analyzed as permethylderivatives by MALDI-TOF MS but, although extensivestatistical analysis was performed, several of the structures wereincorrect, mainly with regard to chain branching and the positionof fucose substitution. Changes in the levels of the fucosylatedtriantennary glycan bearing the sialyl Lewisx epitope (Neu5Ac-a-(2! 3)-Gal-b-(1! 4)-(Fuc-a-(1! 3)-GlcNAc)) have beenobserved in serum glycans from patients with ovarian cancerbutmost changeswere associatedwith acute-phase glycoproteinssuch as a1-acid glycoprotein that respond to inflammation ratherthan from glycoproteins specifically associated with the cancer.In support of this conclusion, Gornik et al. (2007) have foundsimilar changes in serum glycoproteins in patients with acutepancreatitis and sepsis, both inflammatory conditions. Increasedcore a1! 6-fucosylation ofN-glycans has also been observed intype II diabetes (Itoh et al., 2007). Many of the putative serumcancer biomarkers, therefore, appear to be derived frominflammatory glycoproteins whose changes that can be triggeredby several disease states, not just cancer.

B. Congenital Disorders of Glycosylation (CDG)

The use of MALDI-TOF and other types of mass spectrometryfor screening and diagnosis of congenital disorders of glyco-sylation (CDG) has been reviewed (Marklova & Albahri, 2007)and a short description of a method for detecting CDGs withmonitoring by MALDI-TOF MS with emphasis on monitoringtransferrin glycosylation has also appeared (Wada, 2007). In thelatter study, MALDI-TOF analysis of the intact glycoproteindetected peaks at around 78 kDa that lacked one or both sialylatedbiantennary N-glycans. Experimental details have also beenpublished (Wada, 2008b).

C. Other Diseases

A quantitative study of N-glycans released with PNGase F fromserumglycoproteins obtained frompatientswith liver fibrosis hasshown inter- and intra-assay CVs of peak intensity of <17 and<8% respectively illustrating the quantitative nature of MALDI-TOF analysis (Kam et al., 2007). Three glycans were found thatwere positively correlated with the disease and the study wasclaimed to be the first to show the potential value of N-glycanprofiling for identifying liver fibrosis.

In experiments designed to investigate the action of the drug5-fluorouracil (72), used to treat various cancers, S. cerevisiaecells (strain W303-1A) have been treated with the drug andits conjugates with glucose and GlcNAc have been identified by

MALDI-TOF in negative ion mode from THAP. The amountof conjugate detected was directly related to the concentrationof 5-fluorouracil in the culture medium (Gunther Sillero et al.,2007). Ishii, Matsumura, and Toshima (2007) have designed amolecule that selectively binds to and degrades the tumor-associated carbohydrate molecule Gal-b-(1! 3)-GalNAc. Themolecule consists of an anthraquinone linked to a lectin (73). Thelectin specifically binds the carbohydrate, which is then cleavedby radical-induced photo-oxidative cleavage upon irradiationwith UV light. MALDI-TOF analysis was used to characterizethe oxidized products from cyclodextrin used in the developmentof the photoactive molecule. Table 17 lists other studies whereMALDI MS has been used in medical and clinical studies.

XV. GLYCOSYLATION AND OTHER REACTIONMECHANISMS

MALDI analysis is frequently used to characterize the productsof investigations into enzyme action. Such reports are listed inTable 18. In other studies on enzymes, a simple and rapidMALDI-based method for the characterization of substratespecificities of polysaccharide-active enzymes has been devel-oped by Leboeuf et al. (2008) and used to characterize axyloglucan fucosyltransferase and a pectin methyl-esterase.Enzyme reactionmixtures were spotted onto a PVDFmembrane,dried and cleaned by treatment with an ethanol–water mixture.Subsequently, the reaction products were hydrolyzed by specificendoglycanases and the resulting oligosaccharides were directlyanalyzed by MALDI-TOF MS from DHB. The method was saidto be amenable to high-throughput analysis. The interaction ofVicia villosa agglutinin-B4 with glycopeptides with O-linkedGalNAc residues has been investigated by surface plasmonresonance and the affinity was shown to be influenced bythe arrangement of O-glycosylation sites on the peptidePTTTPITTTTK, representing the tandem repeat of MUC2. The



TABLE 17. Use of MALDI MS for examination of glycans in disease1

Disease Medium1 Methods2 Notes Reference Cancer

Breast cancer Serum PNGase F,

TOF/TOF (DHB), N-glycans (per-Me)

Detailed structural analysis of approx 50 high-mannose and complex N-

glycans. Increases in fucose and tetra-antennary glycans in cancer (4 stages)

(Kyselova, et al., 2008)

Breast cancer Serum

PNGase F (in gel), TOF

(DHB), N-glycans,ESI (-ve)

Increase in fucosylated triantennary glycan with disease progression but associated with acute-phase proteins

rather than directly to cancer

(Abd Hamid, et al., 2008)

Breast cancer Cells

PNGase F, TOF/TOF (DHB), glycans (per-Me,

[2H3]-Me)

Development of quantitative method (see text). Glycan composition only

(Kang, et al., 2007)

Breast cancer Serum β-Elimination, FT-

ICR (DHB), complex N-glycans

Significant differences between profiles obtained by MALDI from patients and controls but patient sample too small to

claim biomarkers

(Kirmiz, et al., 2007b)

Colon carcinoma HT29 cells

PNGase F, TOF (DHB) N-glycans

(per-Me), ESI, GC/MS

Significant changes of α-2,3- and α-2,6-sialylation of membrane

glycoproteins between proliferating and differentiated cells.

(Vercoutter-Edouart, et al., 2008)

Colon carcinoma Mouse serum

PNGase F, TOF, Q-TOF (DHB),

complex N-glycans (per-Me)

Induction of acute-phase glycoproteins. Increase in α2→6-Neu5Gc,

substitution by Neu5Ac

(Lin, et al., 2008b)

Colon carcinoma

TIMP-1glycorotein

PNGase F, TOF (DHB, ATT)

complex N-glycans

GlcNAc-transferase V upregulated and resulting glycans promote

invasive/metastatic properties (6-antenna substituted)

(Kim, et al., 2008g)

Colon carcinoma In vitro TOF/TOF, glycans

(Per-Me, 2-AP)

Identification of specific glycosyltransferases with resins

derivatized with nucleotide diphosphates

(Lin, et al., 2008a)

Colorectalcancer

TIMP-1 from plasma

Trypsin, Q-TOF, TOF/TOF (DHB),

Glycopeptides

Only minimal variation found in TIMP-1 N-glycans between cancer and control samples, therefore poor

biomarker for this cancer.

(Thaysen-Andersen, et

al., 2008)

Head and neck tumors Mouse serum

Trypsin, PNGase F, Q-TOF (DHB), MS/MS,

N-glycans (Ph-NH-NH2 derivs)

More than 40 high-mannose, hybrid and complex N-glycans identified. Fucosylated, sialylated biantennary

glycan increased in cancer. Some novel structures proposed.


Hepatocellular carcinoma Serum


N-glycans (per-Me)

Computational methods developed for detecting peptide and glycan

biomarkers of hepatocellular carcinoma and chronic liver disease

(Ressom, et al., 2008b) (Ressom, et al., 2008a)

Hepatocellular carcinoma

α-Fetoprotein L3 from cells

and serum

PNGase F, TOF (DHB), N-glycans

(2-AP)

Changes in activity of GlcNAc transferases III and IV but not V

(Nakagawa, et al., 2008)

Hepatocellular carcinoma Serum

“Enzymatic”, TOF/TOF (DHB),

N-glycans (per-Me)

Development of peak selection algorithm to differentiate hepatocellular

carcinoma and chronic liver disease

(Varghese, et al., 2008)

& HARVEY



Hepatocellular carcinoma Cancer tissue IR-MALDI-o-TOF

(glycerol)Increases in CD75s- and iso-CD75s-

gangliosides observed in cancer (Distler, et al., 2008a)

Leukemia Jurkat cells PNGase F, TOF (DHB), N-glycans

Na+/K+-ATP inhibitors, e.g. digoxin inhibit N-glycan synthesis

(Zavareh, et al., 2008)

Lung cancer Human serum PNGase F, QIT-

TOF (THAP), N-glycans

Method development and identification of differences in core fucosylation of

complex N-glycans in lung cancer

(Ueda, et al., 2007)

Metastaticmelanoma

α3 β1 and αv β3integrin

PNGase F (in gel), TOF

(DHB), N-glycans

From WM9 and WM293 cell lines. Heavily sialylated and fucosylated

glycans plus Gal-GlcNAc extensions

(Kremser, et al., 2008)

Ovarian cancer Serum glycoproteins

PNGase F (in-gel), TOF (DHB), ESI

HPLC (2-AB)

Abundance changes in N-glycans from acute-phase glycoproteins and IgG in

ovarian cancer

(Saldova, et al., 2007)

Ovarian cancer Serum glyco-proteins

By-products of β-elimim., FT-ICR

(DHB), N-glycans

Fibronectin, apolipoprotein B-100 and IgA1 glycosylation altered in cancer

(Li, et al., 2008a)

Ovarian cancer Tumor tissue Trypsin

PNGase F,TOF, N-glycans (per-Me)

Detection of Neu5Gc in human tumor tissue leads to antibody-mediated

inflammation in carcinoma progression

(Hedlund, et al., 2008)

Ovarian cancer Serum β-elimination, FT (2,5-DHAP)

16 Glycans changed in cancer. Claimed to be better biomarkers than CA125

(Leiserowitz, et al., 2008)

Ovarian cancer Plasma β-elimination, FT-

ICR (DHB), Glycans

Mainly method development. Major changes in glycosylation noted but

glycans not identified

(Williams, et al.,

2008a)

Ovarian cancer and lymphoma

AGP from serum

PNGase F, TOF, Q-TOF (DHB), N-

glycans (2-AA)

Changes in fucosylation and branching indices too small for biomarkers but

linear discriminant analysis successful.

(Imre, et al., 2008)

Pancreatic carcinoma Cancer tissue IR-MALDI-o-TOF

(glycerol)Increases in Gb3Cer and iso-CD75s-

gangliosides observed in cancer (Distler, et al., 2008a)

Pancreatic carcinoma

Ribonuclease 1 (Human pancreas/ serum)

PNGase F (in-gel), R-TOF (DHB),

ESI, MS/MS, N-glycans

Increase in core fucosylation of complex N-glycans proposed as

possible biomarkers

(Barrabés, et al., 2007)

Prostate cancer Serum PNGase F, FT-ICR, N-glycans

For detection of potential biomarkers. Changes in high-mannose and

fucosylated biantennary glycans

(de Leoz, et al., 2008)

Prostate cancer PSA and seminal plasma

Lysylendo-peptidase, TOF,

QIT-TOF (DHB), glycopeptides

Mainly biantennary. PSA glycans contained α2→3-linked sialic acid. Glycans in seminal carried α2→3-

linked sialic acids. Possible biomarker

(Tajiri, et al., 2008)

Prostate cancer Haptaglobin from serum

PNGase F, R-TOF, TOF/TOF (DHB),

N-glycans (per-Me)

Haptoglobin levels increased in cancer. Complex N-glycans attached to defined

peptide region showed enhanced branching and antenna fucosylation.

(Fujimura, et al., 2008)

Stomach cancer MKN7 and MKN45 cells

TOF, QIT-TOF (DHB), N-glycans

(2-AA)

Analysis of free complex N-glycans in cytosol. Degraded by cytosolic sialidase. Possible biomarkers

(Ishizuka, et al., 2008)

CDG

CDGII/HEMPAS

Erythrocyte membranes

PNGase F, TOF (CHCA), N-

glycans (per-Me)

Truncated N-glycans found in disease. Explains decreased tomato lectin

binding

(Denecke, et al., 2008)

CDG Ia, CDG IIx

Transferrin and serum α1-


Complex N-glycans separated by yolk immunoglobulins for analysis by (Sturiale, et

al., 2008) antitrypsin N-glycans MALDI-TOF MS




CDG (General) Transferrin

PNGase F (in gel), L-TOF (THAP,

NH4citrate)N-glycans

Heterogenic glycosylation of different glycosylation sites not responsible for complex spot pattern on SDS-PAGE

gels. Due to cysteine oxidation.

(Kleinert, et al., 2007)

CDG II Apolipoprotein C-III (serum)

PNGase F, TOF (DHB), GC/MS, N-glycans (per-Me)

Three patients. Less galactosylation and sialylation of biantennary glycans.

(Bruneel, et al., 2008)

CDG-II Human serum

PNGase F, β-elimination., TOF (DHB), glycans,

GC/MS exoglycosidase

Development of method for examination of both N- and O-linked

glycans

(Faid, et al., 2007)

CDG-IIh or CDG-II/COG8.

Transferrin, human serum

PNGase F, R-TOF (DHB), glycans

(Per-Me)

New CDG caused by mutations in COG8 results in defective sialylation of

O- and complex N-linked glycans

(Kranz, et al., 2007)

CDG-IIh or CDG-II/COG8. Serum

PNGase F (N-linked) β-elimination, R-

TOF (DHB), glycans (Per-Me)

Truncated Cog8 subunit lacking the 76 C-terminal amino acids. N- and O-

glycans contain mild sialylation deficiency.

(Foulquier, et al., 2007)

CDG IIx Transferrin from serum

PNGase F, R-TOF, N-glycans (per-Me)

Patient report. Many truncated complex glycans lacking galactose and sialic

acid

(Nsibu, et al., 2008)

Various Urinary glycans

TOF (DHB), glycans (per-Me),

GC/MS

Development of method for profiling glycoproteinoses

(Faid, et al., 2008)

Unspecified Urine Q-TOF (THAP,

NH4 citrate), Glycans

Low energy CID plus computer-assisted assignment and data base search proposed as most efficient

strategy for rapid ident. of complex carbohydrate in clinical glycomics.

(Vakhrushev, et al., 2008)

Other

Alzheimer’s disease

Mouse commercial

PNGase F, TOF (sinapinic),

glycoproteins

Deglycosylated antibodies effectively induce amyloid β-sequestration without

provoking neuroinflammation

(Takata, et al., 2007)

Bladder reconstruction

MUC2 from bladder mucus

Trypsin, TOF (DHB), O-glycans

Colon tissue, frequently used for this purpose produces much mucin. This

appears to be abnormally glycosylated

(Robbe-Masselot, et

al., 2008)

Cystitis Urine PNGase F, TOF

(DHB), N-glycans(per-Me)

Defective Tamm-Horsfall protein found in patients with interstitial

cystitis

(Parsons, et al., 2007)

Familialtumoral

calcinosis Peptides Intact, TOF,

Glycopeptides Investigation of the role of GalNAc-

transferase T3 in disease

(Kato & Clausen,

2007)

IgA Neuropathy IgA1 from serum Trypsin, TOF Deposition of IgA1 with incomplete O-

linked glycans in hinge region (Iwase &

Hiki, 2008) IgA

nephropathy-like disease

Mouse PNGase F, TOF (DHB), N-glycans

Mice deficient in β-1,4-Gal-transferase develop IgA nephropathy-like disease

(Nishie, et al., 2007)

Liver fibrosis Serum glycoproteins

PNGase F, TOF (s-DHB)

Quantitative MALDI for detection of liver fibrosis biomarkers. Complex

glycans

(Kam, et al., 2007)

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numbers and sites of attached GalNAc residues on thepeptides were confirmed by MALDI-TOF MS (Kato et al.,2008a).

XVI. CARBOHYDRATE SYNTHESIS

Recent reviews containing information on MALDI analysisin carbohydrate synthesis include an article on synthesis ofpolysaccharides via enzymatic polymerization (Kobayashi,2007), a minireview of methods for synthesis of glycans labeledwith stable isotopes (Yamaguchi, 2008) and a review of enzyme-catalyzed reactions on solid surfaces (Laurent, Haddoub, &Flitsch, 2008b). Synthesis with glycosyl transferases is a popularalternative to purely chemical methods; large-scale synthesis ofglycopeptides is frequently performed with a set of enzymesenabling transferases to be reused. Bourgeaux et al. (2007) havedeveloped a system that reduces the number of enzymes fromsix to four. Glycosylated products were analyzed by MALDI-TOF MS.

Unfortunately, many investigators do not report theconditions for recording MALDI spectra in this area. AlthoughDHB is the most popular matrix for simple oligo- and poly-saccharides, some more complicated compounds require differ-ent matrices. Thus, for example, a series of sulfated cyclodextrinderivatives has been synthesized by treatment of the respective

cyclodextrin with a sulfur trioxide-pyridine complex undervarying conditions. The resulting products were heterogeneousmixtures of cyclodextrin sulfates with degrees of sulfation from0-24. The compounds were analyzed initially by MALDI-MS toobtain an estimate of the range of the degree of sulfation in eachsample mixture. Six matrices were used but although these weresaid to give different results, presumably because of sulfate loss,details were not reported. Consequently, elemental analysis wasemployed to determine the average degree of sulfation (Crandallet al., 2007).

A. Glycodendrimers and Glycoclusters

The high molecular weights of glycodendrimers continuesto make this area popular for analysis by MALDI-TOF MS.A review in two parts on glycodendrimers with informationon MALDI analysis has been published by Niederhafner,Sebestık, and Jezek (2008a,b). The reviews together contain593 references.

One of the largest glycopeptides glycodendrimer synthe-sized to date has eight glycopeptides, each bearing a Man3-GlcNAc2 core pentasaccharide coupled via a thioester to aPAMAM core bearing eight amino groups (74). MALDI-TOFanalysis from DHB gave a mass of 29,660 (Ozawa et al.,2007). MALDI-TOF measurements of a cellobiose-polylysine


reticulum α-glucosidase inhibition (by e.g. NBDNJ)

Mental retardation

Serum, fibroblasts

PNGase F, R-TOF (DHB), N-glycans

(per-Me)

Serum N-glycans normal but patients showed mutations in gene encoding oligosaccharide-transferase complex

(Molinari, et al., 2008)

Multiple sclerosis Mice

PNGase F, TOF, N-glycans, (complex)

GlcNAc-branching deficiency may promote T cell-mediated demyelination

and neurodegeneration

(Lee, et al., 2007e)

Osteoarthritis Articular

cartilage in rabbits

PNGase F, TOF, TOF/TOF

(DHB), N-glycans (2-AP)

Sialylation and fucosylation increased 7 days after cruciate ligament transection

(Matsuhashi,et al., 2008)

Pancreatitis and sepsis

Serum glycoproteins

PNGase F (in-gel), TOF (DHB), ESI

(negative ion), HPLC (2-AB)

Increased fucosylation observed in pancreatitis and sepsis

(Gornik, et al., 2007)

Rheumatoid arthritis Hyaluronan TOF (DHB), SEC

Oxidation by seven oxidative systems to model conditions in inflamed joint. Proceeds mainly via hydroxyl radicals

(Soltés, et al., 2007)

Schistosomiasis Urine TOF/TOF, glycans Hex1HexNAc5-7Fuc6-10 glycans as markers for S. mansoni infection

(Robijn, et al., 2008)

Werner syndrome

Human serum IgG

Gas-phasehydrazinolysis,

TOF, N-glycans,ABEE derivs

Patients exhibit aging phenotype for sialylation and galactosylation in outer

arms of IgG oligosaccharides

(Kuroda, et al., 2007)

Lysosomal storage disease

Human leukaemia HL60 cells

Free glycans, TOF (DHB), HPLC, glycans (2-AA)

Free glucosylated high-mannose N-glycans as markers for endoplasmic- (Alonzi, et

al., 2008)

1Human unless otherwise stated.2Format (not all items present): Glycan release method and/or protease, MALDI method (matrix), compounds run (derivative), other

methods.



TABLE 18. Use of MALDI to study the products of enzymes action on carbohydrates

Enzyme Source Methods1 Notes Reference Glycosyltransferases

β-N-Acetyl-glucosaminidases Bombyx mori TOF (DHB) Molecular cloning and expression

of two novel aminidases (Okada, et al.,

2007)

Cyclodextrin glucanotransferase

Pyrococcus furiosus

DSM3638TOF

Enzyme responsible for synthesis of cyclodextrins in hyperthermophiles

(Lee, et al., 2007d)

α-1,3-Fucosyltransferase

Apis mellifera (honey-bee)

Dabsyl glycol-

peptides

Fuc-Ts that transfer fucose to the 3-position of N-linked core

GlcNAc and antenna GlcNAc

(Rendic, et al., 2007b)

1,3-Fucosyltransferase Caenorhabditiselegans

TOF (DHB), glycans

Results suggest that each α1,3-FucT in C. elegans has unique

specificity and expression patterns.

(Nguyen, et al., 2007)

α1-3-Fucosyltrasferase 1 Zebrafish

TOF (DHB), Glycans (2-

AP)

Free Lewis x-biantennary glycans expressed in segmentation period

of zebrafish embryo

(Moriguchi, et al., 2007)

Fucosyltransferase III

Recombinant human in Nicotiana

tabacum L.

TOF Study of N-glycans. Enzyme acted

more like a hydrolase than a transferase

(Lhernould, et al., 2008)

Fucosyltransferase IX Human L-TOF/TOF (DHB)

Able to fucosylated GlcNAc without Neu5Ac in asialo-EPO

(Brito, et al., 2008)

β-(1-3)-Galactosyltransferase

Arabidopsisthaliana

TOF,LC/MS

Identification of galactosyltransferase responsible

for biosynthesis of Lewisa

(Strasser, et al., 2007)

β-(1-3)-Galactosyl-transferase CgtB

Campylobacterjejuni TOF Three variants shown to have

different acceptor specificities (Bernatchez, et

al., 2007) β-(1,3)-

GalactosyltransferasesArabidopsis

thaliana TOF

(CMBT) Characterization of a group of

enzymes (Qu, et al.,

2008)

β-1,3-Galactosyltransferase

Escherichia coliO127

MALDI, ESI

Characterization and application in the synthesis of tumor-associated

T-antigen mimics

(Yi, et al., 2008)

β-(1→4)-Galactosyltransferase

Glycine max Merr (soybean) TOF (DHB) Investigation of chain elongation

of pectic β-(1→4)-galactan(Konishi, et al., 2007)

GalNAc and GlcA transferases (chondroitin polymerase)

Escherichia coli K4 TOF (DHB)

Alternate addition of GalNAc and GlcA to acceptor chondroitin

polysaccharides in the presence of Mn2+ ions.

(Sugiura, et al., 2007)

β-1,2-GlcNAc-transferase Human

L-TOF (DHB), Glycans

Studies with mutants show that Cys-121 has a structural role in

maintaining active site geometry

(Saribas, et al., 2007)

4-α -Glucano-transferase (TAα GT)

Thermus aquaticus TOF (DHB)

Action mode with starch-binding domain on amylose and

amylopectin

(Park, et al., 2007c)

4-α -glucano-transferase (TAαGT)

Sulfolobus sulfataricus

ATCC 35092 TOF (DHB) Catalyses transfer of α1→4-

glucans between molecules (Park, et al.,

2007b)

α(1,6)-Mannosyltransferase

Corynebacterium glutamicum

TOF (DHB), NMR

Identification of enzyme involved in lipomannan biosynthesis

(Mishra, et al., 2007)

Mutant β-(1-4)-Galactosyltransferase Mutant

L-TOF (DHB),

Can transfer 2-keto galactose from UDP derivative opening possibility (Boeggeman,

et al., 2007) Glycans of sensitive test

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Peptide-O-xylosyltransferase

(XT-II) Human TOF Identification of a second form of

the enzyme (Voglmeir, et

al., 2007)

Polyprenol monophospho-

mannose-dependent α(1→6)-

mannosyltransferase

Mycobacteriumsmegmatis

TOF (DHB), ESI, NMR

Use of synthetic α-(1→6)-linked octyl Manp oligomers to probe

substrate specificity

(Tam, et al., 2008)

Various Colonic

carcinoma cell line

TOF(CHCA),

glyco-peptides

Interleukin-4 induces transferases T1, T4 and T7

(Kanoh, et al., 2008)

Various Human TOF (DHB, CHCA)

Lectin domains found on most GalNAc transferases modulate the kinetic properties of the enzymes.

(Wandall, et al., 2007)

Xylogalacturonanxylosyltransferase

Arabidopsisthaliana

R-TOF(DHB) Involved in pectin biosynthesis (Jensen, et al.,

2008)

Xyloglucan xyloglucosyl-

transferase Barley R-TOF/TOF

(DHB)

Catalyses formation of covalent linkages between xyloglycans and β-D-glucans, previously thought to

be non-covalent

(Hrmova, et al., 2007)

α-1,6-xylosyltransferase

Arabidopsisthaliana

TOF (DHB), GC/MS, HPAEC

Involved in xyloglucan biosynthesis. Encodes by XXT5

gene

(Zabotina, et al., 2008)

Xylosetransferase Tropaeolum ajus(Nasturtium)

Linear ion trap

Identification of gene encoding xylosetransferase

(Cocuron, et al., 2007)

Endo-glycosidases

Endo-α -N-acetyl-galactosaminidases

Bifidobacteriumlongum and Clostridiumperfringens

TOF(CHCA) Characterization of two enzymes (Ashida, et al.,

2008)

Endo- -1,4-glucanase Aspergillus niger TOF (DHB) Recombinant expression, purification and characterization

(Master, et al., 2008)

Endo-(1→3)-β -D-glucanase and β-D-

glucosidase

Littorina kurila (mollusc) TOF (DHB)

Characterization of enzyme complex from liver of the marine

mollusk that hydrolyzes laminaran

(Pesentseva, et al., 2008)

Endoglucanase Three different species TOF (DHB) Sensitivity for substituents in

hydrolysis of Me-cellulose (Schagerlöf, et

al., 2007) Endo-α -N-acetyl-galactosaminidase

Enterococcus faecalis TOF (DHB) Novel enzyme. Molecular cloning,

expression, and characterization (Goda, et al.,

2008)

Endo-β -N-acetyl-glucosaminidase

(endo A)

Arthrobacterprotophormiae TOF (DHB)

Glu-173 identified as active site by “chemical rescue” using E173A

mutant with little activity. Restored with sodium azide or formate.

(Fujita, et al., 2007)

Endo-1,4-β -xylanase Trichoderma reesei

L-TOF (DHB)

Immobilized enzyme on membrane cleaved partially hydrolysed

oligoxylose

(Cano & Palet, 2007)

Exo-glycosidases

Amylo-α-1,6-glucosidase

Porcine liver glycogen

debranching enzyme

TOF (DHB), glycans (2-

AP)

Active site mapping using fluorogenic 6-O-α -glucosyl-

maltooligosaccharide

(Yamamoto, et al., 2007a)

α1,2-L-Fucosidase Lilium longiflorum (Ishimizu, et Ident. of novel fucosidase acting TOF,

β

glycans (2-(Lilly) AP) on xyloglucan glycans associated

with endo- β-mannosidase al., 2007)




GH95 α1,2-fucosidase Arabidopsisthaliana

TOF/TOF (DHB)

Ident. of gene encoding fucosidase active on Xyl-Glc poly-saccharides

(Léonard, et al., 2008)

β-Galactosidase (family 42)

Alicyclobacillusacidocaldarius TOF (DHB) Purification and characterization (Di Lauro, et

al., 2008)

β-Glucuronidase (family 79)

Aspergillus niger and Neurospora

crassa TOF

Prop. of enzymes that hydrolyze β-GlcA and 4-O-Me-β -GlcA residues of Ara-Gal-protein

(Konishi, et al., 2008)

PNGase (png-1, F56G4.5)

Caenorhabditiselegans

TOF(THAP)

Invest. of protein disulfide reduction and deglycosylation

(Kato, et al., 2007b)

Other hydrolases N-acetylmuramoyl-L-

alanine amidase B Pseudomonas

aeruginosa L-TOF Production, purification and confirmation of activity

(Scheurwater, et al., 2007)

β-Agarase Agarivorans albus YKW-34 TOF, NMR Purification and characterization (Fu, et al.,

2008b)

Chondroitin hydrolase Caenorhabditiselegans

TOF (DHB), Glycans (2-

AB)

First demonstration of a chondroitin hydrolase in an animal

(Kaneiwa, et al., 2008)

κ-Carrageenase Plocamium

telfairiae (red alga)

TOF, NMR Isolation and activity at various temperatures

(Zhou, et al., 2008a)

Glucuronoxylan xylanohyrdolase

Erwiniachrysanthemi

L-TOF (DHB)

Excellent biocatalyst for making large acidic oligosaccharides

(Vrsanska, et al., 2007)

Hyaluronan lyase Escherichia coli TOF(THAP)

Immobilized metal affinity magnetite for purification of

recombinant hyaluronan lyase from unclarified feedstock

(Yang & Lee, 2007)

Lysozyme (N-acetylmuramide glycanhydrolase)

Tapes japonica TOF Crystal structure. Quaternary

structure and activity modulated by salt concentration

(Goto, et al., 2007)

Maltase-glucoamylase Human

duodenum mucus

TOF Investigation of factors affecting

activity of enzyme for starch digestion

(Quezada-Calvillo, et al.,

2007)

Rhamnogalacturonan lyase

Cotton cotyledons

TOF(THAP/

NO2-cellulose)

Activity in intercellular spaces of cotton cotyledons following

injection of fluorescently-labelled oligo-rhamnogalactuonan

(Naran, et al., 2007)

TreX (iso-amylase) Sulfolobus

solfataricus ATCC 35092

L-TOF (DHB)

Novel iso-amylase possessing the properties of 4-α -glucanotransferase

(Park, et al., 2007b)

Other enzymes acting on sugars 4-α -Glucano-

transferase (TA αGT) Thermus aquaticus TOF (DHB) Action mode with starch-binding

domain on amylose + amylopectin (Park, et al.,

2007c) Chitin oligosaccharide

deacetylase Vibrio cholerae TOF, ESI Characterization and genetics (Li, et al., 2007f)

Cyclic-β -1,2-glucan synthase Various bacteria TOF (DHB) Mechanism for regulating the size

of cyclic periplasmic glucans (Ciocchini, et

al., 2007) Glycogen debranching

enzyme Porcine liver TOF (DHB) Study of substrate specificity of 4- α-glucanotransferase activity

(Watanabe, et al., 2008b)

Heparosan synthases Pasteurellamultocida,

PmHS1, PmHS2

TOF (ATT, THAP)

Expression, activity and use for synthesis

(Sismey-Ragatz, et al.,

2007) 1Format (not all items present): MALDI method (matrix), compounds studied (derivative) other methods.

& HARVEY


dendrimer with a maximum of 24 antennae has given massesbetween 16 and 21 kDa showing that the product lacked from twoto eight antennae (75) (Han, Yoshida, &Uryu, 2007a). In anotherpublication (Han et al., 2007b), the authors report the preparationof further cellobiose-polylysine dendrimers containing 8 and24 antennae. Whereas MALDI-TOF analysis showed completesubstitution of the smallermolecule, themajor ion from the largercompound (m/z 20,009.6) showed the absence of one cellobioseunit. MALDI-TOF was used successfully to analyze the firstand second generation compounds with maltose but it failedwith the maltotriose-based second generation compound (massaround 9 kDa). However the newly developed related method oflaser-induced liquid beam ionization-desorption mass spectrom-etry (LILBID MS; Morgner, Barth, & Brutschy, 2006) produced

a satisfactory negative ion spectrum. More examples are listedin Table 19.

B. Synthesis of Carbohydrate–Protein Conjugates

Again, this is another fertile area for application ofMALDI-TOFMS. For the larger protein complexes, sinapinic, one of theearliestMALDImatrices still appears to be the best. Applicationsare listed in Table 20.

C. Other Syntheses

Syntheses of natural compounds are listed inTable 21 and variousother carbohydrates in Table 22. Papers in which MALDI-MS



& HARVEY


TABLE 19. Use of MALDI MS to measure glycodendrimers

Scaffold Sugar Methods1 Notes Reference

Alkynyl scaffolds High-mannose

(Man4 and Man9)

TOF For binding to DC-SIGN and monoclonal antibody, for HIV vaccines

(Wang, et al., 2008b)

2,2-Bis(hydroxy-methyl)propionic

acid

Mannose, galactose,

lactose

L-TOF (CHCA)

Nanotubes functionalized with glycodendrimers

(Wu, et al., 2008a)

Calix[4]arene

Ribosyl-Me, galactosyl-Meand glucosyl-

methyl fragments

TOF (DHB) Investigation of azide-nitrile

cycloaddition in glycoconjugate chemistry. With propoxytetrazole spacers.

(Dondoni & Marra, 2007)

Calix[4]arene GlcNAcpR-TOF(DHB, CHCA)

As stimulators of NK cell-mediated antitumor immune response. Superior

to the previously tested PAMAM-GlcNAc8

(Krenek, et al., 2007)

Calix[4]arene Neu5Ac TOF (DHB) New inhibitors of hemagglutination and cytopathic effect mediated by BK and

influenza A viruses

(Marra, et al., 2008b)

Calixarene–cyclodextrin conjugates

β-Cyclodextrin TOF (DHB) Construction of tubular compounds (Hocquelet, et al., 2007)

Carbon nanotubes β-D-Galp, α-D-Manp TOF (DHB) Unique scaffold for the multivalent

display of sugars (Gu, et al., 2008)

Carbosilane Lactotriaose TOF Tri-, tetra-, hexa- dodeca-valent. Most measurement by FAB/ESI

(Yamada, et al., 2007a)

Carbosilane Sialyl α(2→3) lactose TOF As influenza hemagglutinin blockers (Oka, et al.,

2008)

Cucurbituril β-Glc, β-Gal, α-Man

TOF Construction of carbohydrate wheels (Kim, et al., 2007d)

Curdlan (linear β-1,3-glucan)

Man, GlcNAc, Lac TOF

From 6-azido-6-deoxycurdlan followed by chemo-selective Cu(I)-catalyzed [3 + 2]-cycloaddition with various carbohydrate

modules having a terminal alkyne

(Hasegawa, et al., 2007)

1,1-Diallyl-2,3,4,5-tetra-

phenylsilole plus trichlorosilane

Globotriaose (Gal α1→4Gal β1→4Glc β1)

TOF Highly luminescent glycocluster (Hatano, et al., 2007)

o-Diamino-benzene Hyaluronan TOF Coupled to the rigid scaffold by

reductive amination (Rele, et al.,

2007)

Di-hydroxy-benzoic acid Galabiose TOF

(CHCA)

Synthesis of multivalent Streptococcus suis adhesion inhibitors by enzymatic cleavage of polygalacturonic acid and

‘click’ conjugation

(Branderhorst, et al., 2008b)

Di-hydroxy-benzoic acid Mannose TOF Attached to aluminium oxide chips to

form microarray for binding studies (Branderhorst, et

al., 2008a)

Fullerene GB3Trisaccharide MALDI Cu catalysed cycloaddition reaction to

attach five oligosaccharides (Isobe, et al.,

2007)

Fullerene Mannose TOF, FAB Product aggregates to form “sugar balls”

(Kato, et al., 2007a)

Gold β-Cyclodextrin Preparation of artifTOF (Li, et al., 2008e) icial supramolecular emyzonan selcitraponan




Gold nanoparticles Globotriose TOF As multivalent probes for shiga-like

toxin (Chien, et al.,

2008)

α-D-Mannose α-D-Mannose TOF

(CHCA), EI, CI

Potential inhibitors of type-1 fimbriae-mediated bacterial adhesion

(Heidecke & Lindhorst, 2007)

Metals (various) Mannose, galactose TOF One-pot synthesis of fluorescent

glycodendrimers (Kikkeri, et al.,

2008) Octane-1,8-

diamide

Benzene-1,3,5-triamide

α-D-Mannose TOF To study binding to mannose receptor (Yin, et al., 2007)

Octapeptide Lewis X MALDI Octapeptide with two tethered Lewisx

epitopes as potential E-selectin ligands (Herzner & Kunz, 2007)

Peptide Mannose TOF (2,3-DHB)

Synthesis in peptide synthesiser. Excellent probe for imaging studies by confocal fluorescence microscopy of mannose-receptor-mediated entry of

into dendritic cells.

(Kantchev, et al., 2008)

Peptide (lysine) Lactose,

dimannose, Lewisa, Lewisx

TOF(CHCA) For dendritic cell targeting (Srinivas, et al.,

2007)

Peptides Gal-GalNAc TOF Sequential segment coupling using N-alkylcysteine-assisted thioesterification

(Ozawa, et al., 2008)

PAMAM plus glycopeptide

(Emmprin peptide)8

(Man3-GlcNAc2)8

TOF Coupled to dendrimer core with eight NH2 groups with thioester

(Ozawa, et al., 2007)

PEG-poly-L-lysine. D-Galactose TOF (DHB) For delivery of drugs (chloroquinine

phosphate) (Agrawal, et al.,

2007) Peptide-PEG nanoribbons. α-D-Man TOF Bacterial cell cluster formation (Lim, et al.,

2007a)

(Ph)6 (branched) D-Mannose TOF Self-assembly into 1-D and 2-D nanostructures

(Ryu, et al., 2007)

Phosphodiester Galactose TOF(THAP)

Combinatorial and automated synthesis of phosphodiester galactosyl cluster on

solid support by click chemistry assisted by microwaves

(Pourceau, et al., 2008)

Phosphodiester oligomers,

pentaerythritol L-Fucose TOF

(THAP) For studying lectin-carbohydrate

interactions (Morvan, et al.,

2007)

Piperazinyl porphyrins Lactose TOF

As potential hepatocyte-selective targeting drugs. Show promising activity in photodynamic therapy.

(Li, et al., 2008b)

Polyethers Galactose,mannose

TOF (DHB, CHCA)

As oligosaccharide mimetics. Mixed Gal and Man

(Elsner, et al., 2007)

Polylysine Acetyl-cellobiose

TOF (s-DHB) Long alkyl spacer to give flexibility (Han, et al.,

2007b) Poly-

(propyleneimine)Maltose and maltotriose TOF (DHB) Synthesis, Structure determination and

Cu(II) complexation(Appelhans, et

al., 2007)

Porphyrin Glucose TOF Retrograde delivery of photosensitizer

(TPPp-O-β GluOH)3 selectively potentiates its photodynamic activity

(Amessou, et al., 2008)

(Niikura, et al., For monitoring cellular chaperones TOF GlcNAc Quantum dots 2007)

& HARVEY


has been used to studymore general reactionswith carbohydratesare listed in Table 23.

XVII. MISCELLANEOUS

MALDI-TOF MS from DHB combined with measurements byUV–vis, 1H-NMR and 2D-NOESYhas been used to characterizethe 1:1 complex between 5,10,15,20-tetrakis[4-(3-pyridinium-propoxy)phenyl]prophyrin tetrakisbromide (TPPOC3Py, 76) andb-CD together with its hydroxypropyl analog in aqueous solution(Qiu et al., 2007). The results show that one of the mesosubstituents of the porphyrin ring deeply penetrates through thecavity of HP-b-CD from the secondary face. Analysis byMALDI-TOF MS of aqueous wood extracts from the preservedSwedish warship Vasa has revealed serious chemical changesdeep in the wood although the surface is less affected.Polyethylene glycol, used as conservation agent, was found tobe partly degraded and low-molecular fragments from hemi-cellulose were found at sites that contained a high iron contentand low pH. The findings imply a catalytic oxidative processand acidic hydrolysis in the wood which presents new problemsfor long-term preservation of the Vasa and possibly othervessels that are preserved in a similar way (Almkvist & Persson,2008).

Other studies include the identification of glycosylatedpeptides obtained by cyanogen bromide cleavage of collagenfrom fetal calf skin (Henkel & Dreisewerd, 2007), theidentification of cellotriose, produced from rapidly growingplant tissue (Johnson et al., 2007) and characterization ofcommercial samples, for example (Castro et al., 2007).

XVIII. CONCLUSIONS

Although, in general, no one mass spectrometric technique candetect all compounds in a mixture (Nordstrom et al., 2008),MALDI-TOF appears to be the best method for profilingmixtures of neutral compounds. Sialylated and sulfated glycans,

however, remain a problem but can be handled after suitabletreatment: derivatization in the case of sialylated glycans and ionpairing for the sulfated compounds. Electrospray ionization,although milder and increasingly popular for glycan analysis,tends to produce ions in different charge states and also causesome fragmentation. MALDI imaging, the use of glycan arraysand ion-mobility mass spectrometry have emerged as the areasshowing greatest technological development; ion mobility, inparticular, appears to offer much for general glycan analysis,particularly following the introduction of commercially availableinstruments. It is anticipated that these techniques will be usedmuchmore extensively in the future and will feature prominentlyin the next review in this series.

XIX. ABBREVIATIONS

2-AA 2-aminobenzoic acidAA-Ac 3-(acetylamino)-6-aminoacridine2-AB 2-aminobenzamideABEE aminobenzoic acid ethyl esterADA 4,40-azodianilineAFP a-fetoproteinAGE advanced glycation end productsAMAC aminoacridoneAMC 7-aminomethylcoumarinANTS 8-aminonaphthalene-1,3,6-trisulfonic acidaoWR Na-((aminooxy)acetyl)tryptophanylarginineAP aminopyrazine2-AP 2-aminopyridineAP-MALDI atmospheric pressure MALDIAQ aminoquinolineAra arabinoseAraN arabinosamineArg arginineAsp aspartic acidAsn asparagineATT 6-azo-2-thiothymineBac bacillosamine (2,4-diamino-2,4,6-trideoxy-D-

glucose)


meso-Tetra(penta-fluorophenyl)-

porphyrin Glucose, xylose MALDI Combinatoral library for production of

photodynamic therapeutics (Samaroo, et al.,

2007)

Tris(2-ethylamono)amine

-polylysine Cellobiose TOF Long alkyl spacer to give flexibility (Han, et al.,

2007a)

Tris(hydroxy-methyl)amino-

methaneMannose TOF (DHB,

CHCA), ESI Designed to serve as mimetics of

glycocalyx constituents. (Shaikh, et al.,

2008)

3-(2-(2-(3-(trityl-amino)-propoxy)-ethoxy)-ethoxy)-

propylamine

GlcNAc MALDI Ligands for the plant lectin wheat germ agglutinin (WGA)

(Maierhofer, et al., 2007)

Various Mannose TOF Man4 to Glc. Coupled with squaric acid links to various scaffolds

(Sperling, et al., 2007)




TABLE 20. Use of MALDI MS to measure carbohydrate–protein conjugates

Carbohydrate-protein conjugates Sugar Protein Method1 Notes Reference

O-Acetylatedsialoglycosides HSA TOF Chemoenzymatic synthesis (Yu, et al.,

2007b) Bacopaside (steroid

glycoside) BSA TOF(sinapinic)

Average of nine attached glycosides (periodate reaction)

(Phrompittayarat, et al., 2007a)

Capsular polysaccharide fragments from Streptococcus pneumoniae

Diphtheria toxoid

(CRM197)

TOF(sinapinic)

Determination of smallest structure (Gal-Glc-(Gal)GlcNAc) that evokes

opsonophagocytic antibodies against S. pneumoniae type 14

(Safari, et al., 2008)

β-D-GlcpNAc3S-(1→3)-α -L-Fucp

BSA TOF

To mimic carbohydrate in proteoglycan-like macromolecular

complex from Microciona prolifera(sponge)

(Kamerling & Carvalho de Souza, 2008)

Glycolipoprotein BSA TOF Cholesterol-Gal-C16-acid terminating in CHO group plus BSA

(Pozsgay & Kubler-Kielb,

2007)

GM1 tetrasaccharide KLH TOF(CHCA) MALDI to analyse carbohydrate (Yoshikawa, et

al., 2008b)

GM3 plus others with Neu5Gc HSA TOF

Study of diversity of anti-Neu5Gc antibodies in humans and implications

for disease

(Padler-Karavani, et al.,

2008)

Lactose BSA SELDI-TOF Comparison of squarate reagents. Di-Me-squarate most convenient

(Hou, et al., 2008b)

Lactose and glycan from asialo-GM1 BSA TOF

(sinapinic) To study multivalent binding of ricin (Blome &

Schengrund, 2008)

Maltotriose Quantum

dots, Texas red BSA

TOF Readily transported into the nucleus of digitonin-permeabilized HeLa cells

(Niikura, et al., 2008)

Mannan fragment from Candida

albicansBSA L-TOF

(DHB) Penta-mannan synthesised and conjugated with squaric acid

(Karelin, et al., 2007)

β-1,2-mannan disaccharide on

glucose core

Tetanus toxoid, BSA

TOF(sinapinic,

DHB) Synthesis and immunochemical studies (Wu, et al.,

2007c)

β-(1→2)-Mannan trisaccharides

BSA,tetanus toxoid

TOF(sinapinic)

Glycosidic oxygen replaced by sulfur. Adipic acid link

(Wu, et al., 2008b)

Mono- plus di-Me blood group H

antigenBSA TOF (DHB)

O-Methylated glycans from Toxocarasp. shown to be specific antibody

targets

(Schabussova, et al., 2007)

O-Specificpolysaccharides from Bordetella

bronchiseptica andB. parapertussis

BSA TOF(sinapinic)

Preparation of saccharide, development of new conjugation procedures, and

immunological characterization of the conjugates

(Kubler-Kielb, et al., 2008)

3-Phenoxybenzyl alcohol glucuronide BSA TOF

(sinapinic)

To develop immunoassay for detection of pyrethroid exposure by measuring

metabolite in urine

(Kim, et al., 2007a)

Polysaccharide from Alloiococcus otitidis BSA L-TOF

(sinapinic) Conjugate contained up to seven

sugars. Bacterium causes otitis media. (Arar, et al.,

2008)

& HARVEY


BSA bovine serum albuminCD cyclodextrin or circular dichroismCDG congenital disorder of glycosylationCE capillary electrophoresisCEC capillary electrochromatographyCer ceramideCG chorionic gonadotropinCGE capillary-gel electrophoresisCHCA a-cyano-4-hydroxycinnamic acidCHIQ 1-chloro-4-hydroxyisoquinolineCHO Chinese hamster ovaryCI chemical ionizationCID collision-induced dissociation (decomposition)Cit citrateCMBT 5-chloro-2-mercaptobenzothiazoleCNT carbon nanotubesCon A concanavalin ACTID chemically targeted identificationCV coefficient of variationCZE capillary zone electrophoresisDa DaltonDC-SIGN dendritic cell-specific intercellular adhesion

molecule-3-grabbing non-integrinDCTB 2-[4-tert-butylphenyl-2-methylprop-2-enylidene]-

malonitrileDESI desorption electrospray ionizationDHAP dihydroxyacetophenoneDHB dihydroxybenzoic acidDMA N,N-dimethylanilineDMF dimethylformamideDMSO dimethylsulfoxideDNA deoxyribonucleic acidDP degree of polymerizationDTT dithiothreitolEC enzyme commissionEDTA ethylenediaminetetraacetic acidEI electron impactEPO erythropoietinEPS extracellular polysaccharideESI electrospray ionization

f (as in Galf) furanose form of sugar ringFAB fast atom bombardmentFmoc 9-fluorenylmethoxycarbonylFru fructoseFT Fourier transformFuc fucoseFucNAc N-acetylfucoseamineG2CHCA 1,1,3,3-tetramethylguanidinium salt of CHCAG3CA TMG salt of p-coumaric acidGAG glycosaminoglycanGal galactoseGALDI graphite-assisted laser desorption/ionizationGalNAc N-acetylgalactosamineGC/MS gas chromatography/mass spectrometryGlc glucoseGLC gas–liquid chromatographyGlcA glucuronic acidGlcN glucosamineGlcNAc N-acetylglucosamineGly glycineGPI glycosylphosphatidylinositolGSL glycosphingolipidHABA 2-(40hydroxyphenyl)azobenzoic acidHEK human embryonic kidneyHEMPAS hereditary erythroblastic multinuclearity with

positive acidified serum lysis testHep heptoseHex hexoseHexA hexuronic acidHexN hexosamineHexNAc N-acetylhexosamineHFBA heptafluorobutyric acidHILIC hydrophilic interaction chromatographyHIQ hydroxyisoquinolineHIV human immunodefficiency virusHLA human lymphocyte antigenHMWDOM high molecular weight dissolved organic matterHPAEC high-performance anion exchange chromato-

graphyHPLC high performance liquid chromatography


Polysaccharides including chemically

tagged

Ovalbumin, IgG TOF

Mutant and wild-type glycosyl transferases used to transfer sugars

GlcNAc in glycoproteins

(Qasba, et al., 2008)

Sialic acid HSA TOF Multivalent Neu5Ac conjugates inhibit

adenovirus type 37 from infecting human corneal epithelia

(Johansson, et al., 2007)

6-Sialyllactose Ovalbumin TOF For monitoring influenza virus hemagglutinin

(Mandenius, et al., 2008)

2,3-Sialyl-T antigen (Neu5Ac-Gal-

GlcNAc) BSA TOF Up to nine glycohexadeca-peptides

attached to BSA (Wittrock, et al.,

2007)


(bacopaside 1) BSA TOF

(sinapinic) For producing polyclonal antibodies for

ELISA assay (Phrompittayarat,

et al., 2007b)

1Format: MALDI method (matrix).



TABLE 21. Use of MALDI MS for monitoring products of synthetic reactions

Carbohydrate Methods1 Synthetic methods and Comments Reference Monosaccharides

Bridged tetrazoles L-TOF (DHB, CHCA)

Unexpected reaction from intramolecular 1,3-dipolar cycloaddition of 1,2-O-cyano-

alkylidene derivatives of 3-azido-3-deoxy-D-allose

(Worch & Wittmann, 2008)

6 α-Carba-β -D-fructopyranose MALDI-

FTMS (no matrix)

Synthesised by ring-closing metathesis on 2-C-hydroxymethyl-L-erythrose acetonide

(Totokotsopoulos, et al., 2008)

Chain-extended Se, S, and N analogues of the glucosidase

inhibitor salacinol MALDI For structure-activity studies (Liu, et al., 2007a)

1-Epi-valienamine TOF(THAP)

Cyclohexene -part of the structure of acarbose

(Cumpstey, et al., 2008)

Fluorescent fructose derivatives TOF Fluorophores 7-nitro-1,2,3-benzadiazole

(NBD) and Cy5.5 for imaging breast cancer cells

(Levi, et al., 2007)

Fructose-fused γ-butyrolactones and lactams TOF (DHB) As GABA receptor ligands (Araújo, et al.,

2008) Peracetylated C-glycoside

ketones R-TOF(DHB) For EI fragmentation studies (Price, et al., 2008)

Me-3,4-di-O-Ac-1,5-anhydro-2-deoxy-D-arabino-hex-1-

enopyranuronate TOF For X-ray diffraction and NMR study (Liberek, et al.,

2007)

Me (Me 4-O-Ac-3-azido-2,3-dideoxy-a/b-D-arabino- and -

a/b-D-ribo-HexpATOF Synthesis and geometry (Tuwalska, et al.,

2008a)

Methyl 3-amino-2,3-dideoxyhexopyranosidic acids TOF Synthesis and conformational analysis (Tuwalska, et al.,

2008b)

β-D-Rhamnopyranosides TOF (DHB) 2-Naphthylmethyl ether to make a temporary linkage as a mixed acetal (Lee, et al., 2008c)

S-Alkylated sulfonium ions TOF (DHB) Alkyl group on thiopentoses (Mohan, et al., 2007)

Sialic acid azide TOF (DHB) Study into the mechanism of action of Trypanosoma cruzi trans-sialidase

(Damager, et al., 2008)

Sulfonic acid analogues of N-acetylneuraminic acid TOF First synthesis. from 1-thio-L-fucoside

derivatives (Szabó, et al.,

2008) Oligosaccharides

α-Acarviosinyl-(1-9)-3-α -D-glucopyranosylpropen TOF From acarviosine-glucose and 3-α-D-

glucopyranosylpropen (Lee, et al., 2008b)

Arabinofuranoside glycodynamers TOF “Dynamic” molecules by replacement of

glycosidic link with oxime (Ruff & Lehn,

2007)

Arabinoxylobiose TOF/TOF (DHB)

From rye xylan by cleavage with A. aculeatus endo-1,4-β -D-xylanase

(Rantanen, et al., 2007)

α-Linked 2-deoxyglucosides TOF (DHB) Prepared by employing a glycosyl donor

having a participating (S)-(phenylthiomethyl)benzyl moiety at C-6

(Park, et al., 2008b)

C-9 Acetylated sialosides TOF (DHB) Chemo-enzymatic synthesis (Rauvolfova, et al., 2008)

Aminoethyl-β -D-Galp-(1→4)- β-D-Glcp

TOF Intermediate in the synthesis of Galp-(1→3)- α-D-GalpNAc (T-antigen). For

study of lectin interaction

(Murthy & Jayaraman, 2008)

& HARVEY



Bicyclic amino sugars TOF (DHB) Related to nojirimycin. With cyclic carbamate, urea, guanidine.

(Cipolla, et al., 2007)

Carbohydrate antigens Gb-3 and Globo-H MALDI Automated solid-phase synthesis (Werz, et al.,

2007b)

[2H8]-Cellobiose MALDI (DHB) Synthesis from D-glucose (Zhang & Vasella,

2007)

Chitin/chitosan oligomers TOF (DHB), ESI

From N-deacylated chitosan by partial re-N-acetylation

(Trombotto, et al., 2008)

Chitooligosaccharides TOF/TOF (DHB)

(DHB)

Hydrolysis of high molecular weight chitosan with endochitanase from

Serratia marcescens. For inhibition of family 18 chitinase

(Cederkvist, et al., 2008)

Chitooligosaccharides TOF From N-deacylated chitosan

(commercial). Effect on growth of phytopathogenic fungi

(Oliveira, et al., 2008)

Chitosan TOF (2,4-DHB)

From lobster shells, Used to protect rice (Oryza sativa L.) seeds against attack by

Pyricularia grisea (Cooke) Sacc.

(Rodríguez, et al., 2007)

C-Linked neuraminic acid disaccharide TOF Building block for the synthesis of C-

analogues of polysialic acids (Kim, et al., 2008b)

C-Linked sialosides TOF (DHB,

tri-OH-anthracene)

Use of cross metathesis. (Meinke & Thiem, 2008)

CH2-Linked α(2,3)sialylgalactose analogue

TOF(CHCA)

Synthesized using an Ireland-Claisen rearrangement

(Watanabe, et al., 2008a)

Chitooligomers Reduction of oligomers produced by enzymolysis to prevent browning (Qin, et al., 2008)

Decarboxylated sialic acid dimers TOF By-products of 2,3-dehydro-N-acetyl

neuraminic acid synthesis (Horn, et al., 2008)

Diblock co-oligomers of tri-O-methylated and unmodified

cello-oligosaccharides

R-TOF(DHB) Carbohydrate-based nonionic surfactants (Kamitakahara, et

al., 2007)

Di- and tri-saccharide mimics with amino bridges TOF (DHB) NH Link between non-anomeric positions (Neumann, et al.,

2007)

Gal-β -(1→4)-GlcNAc TOF,

glycans (2-AP)

Development of fluorescence assay for glycosyltransferase reactions with xanthene ZnII-Dpa chemosensor

(Kohira, et al., 2008)

Galactose-containing trehalose analogue disaccharide TOF Enzymatic synthesis, inhibitory effects on

several disaccharidase activities (Kim, et al., 2007b)

1,4-Glucans TOF Synthesis by ring-opening of cyclodextrins with TiCl4

(Bösch & Mischnick, 2007)

Linear and branched oligolactosamines TOF (DHB) As galectin ligands (Severov, et al.,

2007) α(1→6)-Linked octamannan

fluorescent probe TOF From glycosyl fluoride donors and thioglycosyl acceptors

(Aqueel, et al., 2008)

Maltose and maltotriose derivatives

TOF (DHB), FAB

As potential inhibitors of maltose-binding protein

(Malik, et al., 2008)

(1→2)(1→6)-Mannotrioside mimic TOF Central mannose replaced with trans-

diaxial cyclohexanediol (Mari, et al., 2007)

Neoagarose-oligosaccharides TOF (DHB) Enzymatic cleavage of agarose with agarase A or B. (Li, et al., 2007c)

Oligochitosan TOF Enzymatic hydrolysis of chitosan (Zhao, et al., 2007b)

Oligomannans MALDI Regioselective synthesis of α-(3-6) and (Jayaprakash, et al., 2007) α-(2-6) mannan arrays

TOF




3-O-Me-glucose-containing TOF Convergent synthesis (Hsu, et al., 2007c) 6-O-Me-glucose-containing

oligosaccharides TOF Synthesis from cyclodextrins (Meppen, et al., 2007)

Palayinose-containing oligosaccharides TOF

Glucosyl transfer from β -D-Glc-1-PO4 to palatinose with Thermo-anaerobacter

brockii kojibiose phosphorylase.

(Takahashi, et al., 2007)

Pentasaccharide PI-88 MALDI One-pot glycosylations with catalytic activation

(Valerio, et al., 2008)

Penta- and hexa-saccharide derivatives

TOF(CHCA)

(β-D-Glcp-(1→6))4 and 5-(1→4)-α-D-ManpOMe (Liu, et al., 2008a)

Phytoalexin-elicitor heptaglucoside TOF

Stereoselective tris-glycosylation to introduce β-(1→3)-branches into

gentiotetraose (Son, et al., 2008)

Sialyl Lewisx MALDI Neu5Ac-α -(2→3)-Gal building block (Hanashima, et al., 2007)

Sialylated pentasaccharides MALDI, ESI

Related to avian and human influenza virus infection

(Hanashima & Seeberger, 2007)

Sulfhydryl-containing ethylene-glycol-derivatizedoligosaccharides

TOF For conjugation with ovalbumin for targeting to dendritic cells

(Adams, et al., 2008)

Thermo and acid stable TOF (DHB) Synthesised with dextransucrase with high levels of sucrose (Seo, et al., 2007a)

Various TOF Use of n-pentenylorthoesters having

manno-, gluco- or galacto- configurations in regioselective glycosylations

(Jayaprakash & Fraser-Rei, 2007)

Xylo-gluco-oligosaccharides TOF As probes for α-xylosyltransferases (Fauré, et al., 2007)

Xylooligosaccharides TOF Use of Geobacillus stearothermophilusGH52 β-xylosidase

(Ben-David, et al., 2007)

α-D-Xylosylated maltooligosaccharides TOF (DHB) Enzymatic synthesis by phosphorylase-

catalyzed xylosylation(Nawaji, et al.,

2008b)

β-D-Xylp-(1→4)-α -L-Araf-(1→3)-β-D-Xylp-(1→4)-β -D-

Xylp-(1→4)-β -D-Xylp TOF

By hydrolysis of destarched wheat bran. Study of complex between sugar and Thermobacillus xylanilyticus GH-51

arabinofuranosidase

(Paës, et al., 2008)

Polysaccharides

1-Allyloxy-2-hydroxypropyl starch

R-TOF(DHB)

From maize starch plus allyl glycidyl ether. MALDI after digestion with

pullulanase, α-amylase and amyloglucosidase

(Huijbrechts, et al., 2007)

Aminoethyl chitooligosaccharide TOF (DHB) Inhibitition of activity of angiotensin

converting enzyme (Ngo, et al., 2008)

Branched polyglucans L-TOF (DHB)

Synthesised by the tandem action of potato phosphorylase and Deinococcus

geothermalis glycogen branching enzyme

(van der Vlist, et al., 2008)

Cellulose TOF Synthesis from cellobiose (Egusa, et al., 2007)

Cellulose triacetate oligomers TOF Oligomers prepared by pivalolysis. Solubility on dense CO2 evalulated (Hong, et al., 2008)

2,6-Di-O-methyl celluloses L-TOF (DHB)

Synthesised from celluloses, To unravel structure-property relationships (Kamitakahara, et al., 2008b)

(Kamitakahara, et al., 2008a)

Highly branched polysaccharides from sugar TOF (DHB)

Per-Me

Thermal powder-to powder polycondensation. Reaction mechanism (Kanazawa, et al.,

2007) artceps IDLAM morf decuded sediroulf

& HARVEY



α-(1→4)-Linked 6-O-methyl-D-glucose TOF From ring-opened cyclodextrins (Cheon, et al.,

2007a) Sialic acid-containing

polysaccharides TOF By sialyltransferase-catalyzed block transfer

(Muthana, et al., 2007)

N-Substituted trimethyl chitosan MALDI MALDI to determine composition of

starting chitosan 1 (Rúnarsson, et al.,

2008) Unnatural hybrid polysaccharides TOF Enzymatic polymerization catalyzed by a

glycoside hydrolase.(Ohmae, et al.,

2007)

Xyloglucans MALDI Use of mutated aspen xyloglucan endo-transglycosylase (Piens, et al., 2007)

Glycosaminoglycans and related compounds

Betaglycan from linkage region TOF First synthesis of the tetraosyl hexapeptide (Tamura, et al., 2007)

Chondroitin oligosaccharides

R-TOF(DHB),

TOF/TOF, MS/MS

Sequential synthesis by immobilized chondroitin polymerase mutants

(Sugiura, et al., 2008a)

Chondroitin sulfate E octasaccharide TOF, ESI Synthesis from an acetamide-type

disaccharide unit (Tamura, et al.,

2008)

Heparin fragments L-TOF (Caffeic

acid)

Stimulation of hepatocyte growth factor production. Analysed as (RG)19R

(Sakiyama, et al., 2007)

Hyaluronan derivatives TOF (DHB) Hyaluronidase-catalysed copolymerization

(Ochiai, et al., 2007a)

Hyaluronan oligomers L-TOF (CHCA)

Enzymatic digestion (bovine testicular hyaluronidase) of HA 1000.

(Ibrahim, et al., 2007)

Hybrid GAGs TOF (DHB), Nafion plate

Hyaluronan-chondroitin and hyaluronan-chondroitin 4-sulfate synthesized via

enzymatic copolymerization catalyzed by hyaluronidase

(Ochiai, et al., 2007b)

Tetrasaccharide substrate for heparanase

MALDI (DHB)

ΔHexA(2S)-GlcN(NS,6S)-GlcA-GlcN(NS,6S)

(Chen, et al., 2008b)

Uronic acid building blocks for heparin oligosaccharides MALDI Synthesis by stereoselective elongation of

thio-acetal protected dialdehydes (Adibekian, et al.,

2007) N-linked glycans

13C-Neu5Ac-labelled biantennary glycans MALDI Synthesised with [1,2,3,10,11-13C]-CMP-

β-Neu5Ac (Macnaughtan, et

al., 2008)

HexNAc-(Pent)HexA-(Pent)Hex-Hex-HexNAc-Asn

QIT-TOF (DHB)

N-Glycan from Pyrococcus furiosus. New motif (DxxK) found in STT3 g-

transferase. Binds to pyrophosphate (Igura, et al., 2008)

High-mannose glycans and GlcNAc1 analogues, 2-PA

derivatives TOF To develop HPLC system for analysis of

GlcNAc1 compounds from cytosol (Suzuki, et al.,

2008)

Hybrid N-glycan: Man5GlcNAc3

TOF (DHB) Synthesis from Man5GlcNAc2 using GlcNAc-transferase I

(Chen, et al., 2008d)

N-glycan from Campylobacter jejuni. TOF (DHB)

Chemical synthesis of (GalNAc-α(1→4)-GalNAc-α (1→4)-[Glc-β (1→3)]GalNAc-

α(1→4)-GalNAc-α (1→4)-GalNAc- α(1→3)-Bac

(Amin, et al., 2007)

Pig-derived N-glycansimmobilized on 4-

hydrazinobenzoic acid (HBA)-functionalized beads

TOF For binding to and extraction of proteins involved in host-graft rejection (Kim, et al., 2008f)




α-(2,3)-Sialylated core-Fuc-biantennary N-glycan MALDI Pre-activation-based one-pot synthesis

from three components (Sun, et al., 2008a)

Glycopeptides/Glycoproteins

Biantennary glycan plus CTLA-4 fragment (113-150) TOF (DHB)

Underivatized glycan. Side reactions avoided by using low concentrations of

reagents.

(Yamamoto, et al., 2007b)

Biotinylatedoligomannosylpeptoids To study binding to concanavalin A TOF (Yuasa, et al.,

2007) C-Linked antifreeze

glycopeptides TOF (DHB) Study of hydration for inhibition ice recrystallization

(Czechura, et al., 2008)

C-Terminal neoglycopeptidylureas TOF

Incorporation of urea moiety between sugars and peptides employing Curtius

rearrangement

(Sureshbabu, et al., 2008)

TOF Collagen type II analogues

Studies on the interactions in the ternary Aq/glycopeptide/T-cell receptor complexes that activate T cells in

autoimmune arthritis

(Andersson, et al., 2007)

TOF (DHB) Core 4 glycopeptide Benzyl protection. Microwave irradiation to dechlorinate Cl3CO (Ueki, et al., 2007)

Core fucosylated and bisected glycans

L-TOF (sinapinic)

Neoglycoproteins to study influence of fucose or bisect

(André, et al., 2007b)

(Glycans)4 and (amino acids)4on cyclopentapeptide TOF

Sugars = αMan, αGalNAc, βLac, and αFuc; amino acids = Lys, Asp, Tyr, Phe,

and Ile

(Duléry, et al., 2008)

C-Glycosylmethyl pyridylalanines Hantzsch-type three-component synthesis TOF (Dondoni, et al.,

2007)

TOF (DHB) A-Dystroglycan mucin domain To determine which O-linked site in 15-mer peptide is glycosylated

(Breloy, et al., 2008)

Glycopeptide library R-TOF(CHCA)

To assess the inhibitory potency of galectin ligands

(Maljaars, et al., 2008)

TOF (DHB) Glycopeptides from CD52 From Fmoc amino acids and glycan asparagine conjugates

(Swarts, et al., 2008)

α- and β-C-glucopyranosyl serines

TOF(CHCA)

Synthesis of both anomers from common intermediate

(Nolen, et al., 2008)

C-Glycosyl amino acids TOF(CHCA)

Prepared by D- and L-proline-catalyzed α -amination of C-glycosylalkyl aldehydes

using dibenzyl azodicarboxylate

(Nuzzi, et al., 2008)

C-Glycosyl α-amino acids TOF Prepared by click azide-nitrile cycloaddition

(Aldhoun, et al., 2008)

Glycosylated 3 peptides MALDI (CHCA)

Capable of folding into the 314-helicalconformation in water

(Norgren & Arvidsson, 2008)

TOF Helical glycopeptides Comparison with random coil glycopeptides for binding to cholera toxin

(Liu & Kiick, 2008)

Heptapeptide with Man3GlcNAc2

TOF Prepared from IgG Fc fragment by proteolysis and glycolysis to study

binding to Fbs1 lectin.

(Yamaguchi, et al., 2007)

FT-MALDI Lipo-neo-glycopeptides Neoglycosylation of methoxyamine-appended vancomycin aglycon

(Griffith, et al., 2007)

O-Mannosylated peptides (fluorescently labelled) TOF, ESI As components for synthetic vaccines:

comparison of two synthetic strategies(Brimble, et al.,

2008)

MUC1 Glycopeptides L-, R-TOF (DHB)

Identification of cancer-specific glycopeptide (Tarp, et al., 2007)

TOF (DHB) MUC2, MUC6 glycopeptides. Core 3- and Core 6-glycans. FMOC-protected Ser/Thr saccharide.

(Nakahara, et al., 2007)

β

& HARVEY



MALDI MUC4 sialylglycopeptide Sialylation using 5-acetamido-neuraminamide donors

(Okamoto, et al., 2008)

TOF Neo-glycopeptides Importance of sugar core conformation in oxime-linked glycoprobes for interaction

studies

(Jiménez-Castells, et al., 2008)

Podoplanin O-linked glycopeptides

R-TOF(DHB)

To study binding to C-type lectin-like receptor CLEC-2

(Kato, et al., 2008b)

Serogroup A polysaccharide-peptide conjugates TOF To develop vaccine against meningococci

of serogroup A (Filatova, et al.,

2008)

TOF (DHB) Tumor-associated MUC-1 Investigation into the delivery of tumor-

associated glycopeptides to HLA Class II compartments

(Napoletano, et al., 2007)

Vascular endothelial growth factor D inhibitors

TOF (DHB, ATT)

Tetra- plus penta-peptides with two substituted glucose (Haag, et al., 2007)

Peptidoglycans

Proteoglycan core structures TOF/TOF, LIFT (DHB) Synthesis from 1,6-anhydro-β -lactose (Shimawaki, et al.,

2007) Glycosphingolipids

Aplysinella rhax (marine sponge) analogues TOF Synthesis and structure-activity

relationships. Unique structure (Hada, et al., 2007)

CF2 (Hirai, et al., 2007) Stereocontrolled synthesis TOF -linked ganglioside CM4

Q-TOF Clarhamnoside From marine sponge Agela clathrodes. 1st

total synthesis. Tetra-saccharide (Ding, et al., 2007)

MALDI Fluorinated GM4 analogue Fluorine in 2-, 4- and 6-positions of galactose

(Kasuya, et al., 2007)

Glycosphingolipids from earthworm (Pheretima

hilgendorfi)TOF, FAB

Syntheses and biological activities of amphoteric glycolipids containing a

phosphocholine residue

(Hada, et al., 2008b)

Glycosphingolipids from Mucorhiemalis TOF, FAB Synthesis of compounds with Gal-α-

(1→6)-Gal linkages (Hada, et al.,

2008a)

GM1, GM2, GM3 probes TOF(CHCA)

Ceramide replaced by glucose. For probes for microarray

(Imamura, et al., 2008)

Lyso-GM3, monomer, dimer and multimer TOF Synthesis and effect on epidermal growth

factor-induced receptor tyrosine kinase(Murozuka, et al.,

2007) 6′-NeuAc-GM2 and α-GalNAc-

GM2TOF

(CHCA) To elucidate mechanism underlying

hydrolysis of GalNAcβ1→4Gal(Komori, et al.,

2008)

N-linked-ceramide analogues TOF N-linked glycans coupled to amino-ceramide by reductive amination (Yoon, et al., 2007)

7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) labelled glycosphingolipids

TOF (DHB) For visualization of Lewisx

glycosphingolipids (Gege, et al., 2008)

6-O-sulfo-sialylparagloboside TOF(CHCA) Total chemical synthesis. As glycoprobe (Yamaguchi, et al.,

2008a) GPI Anchors

MALDI CD52 Antigen Convergent synthesis from trimannose and phospholipidated di-sacc. (Wu & Guo, 2007)

CD52 and derivatives from sperm TOF Found to interact with pore-forming

bacterial toxin CAMP (Wu, et al., 2008c)

Synthetic analogues TOF(sinapinic)

Synthesis and behaviour in supported lipid bilayers

(Paulick, et al., 2007)

Carbohydrates from plantsRhamnogalacturonan II (B

chain) tetrasaccharide TOF (DHB) Synthesis and immunological properties (Rao, et al., 2008)




8-Azidooctyl derivatives of O-chain from E. coli O9a MALDI Plus analogue with methyl group on O-3

of the distal Manp residue (Hou, et al., 2008a)

Carbohydrates from Mycobacterium smegmatis TOF Shift in UV absorption when bound to

tetraenoic fatty acids (Cheon, et al.,

2007b) Capsular polysaccharide analogues from Neisseria

meningititids A To develop glycoconjugate vaccine FT-ICR (Torres-Sanchez, et

al., 2007)

Capsular polysaccharide components from Cryptococcus

neoformansTOF

Variant synthetic pathway to GlcA-containing di- and trisaccharide thioglycoside building blocks

(Vesely, et al., 2008)

Common tetrasaccharide motif of LPS inner core structures

from Haemophilus influenzae

TOF(THAP)

L-glycero- -D-manno-Hepp-(1→2)-(6-O-NH2EtPO4-L-glycero-α-D-manno-Hepp)-(1→3)-[ β-D-Glcp-(1→4)]-L-glycero-α -D-

manno-Hep

(Mannerstedt, et al., 2008)

Docosanasaccharide arabinan from Mycobacterium

tuberculosis

TOF (DHB, IAA,

DCTB)

To probe biosynthesis of arabinan-containing polysaccharides. Highest

MALDI mass = 7785 (Joe, et al., 2007)

Dodecasaccharidyl lipomannan from Mycobacteria MALDI Glycan component prepared from D-

mannose (Fraser-Reid, et al.,

2008) Hexasaccharide repeat from

Bacillus anthracis MALDI With n-pentenyl handle at reducing terminus

(Oberli, et al., 2008)

Lipid A from Neisseriameningitidis TOF/TOF To investigate effect of acyl groups on

inflammatory props. (Zhang, et al.,

2008f) Lipid A derivative from E. coli

andS. typhimurium For study of cytokine secretion TOF (Zhang, et al., 2007f)

Lipid A modified by an ether-linked lipid from Rhizobium

sin-1TOF Synthesis, agonistic and antagonistic

properties(Vasan, et al.,

2007)

Nonasaccharide subunits of the atypical O-antigen

polysaccharide from Helicobacter pylori strains

TOF Synthesis by dimerization and

trimerization of suitably protected trisaccharide repeating unit

(Fulse, et al., 2007)

Pentasaccharide repeat from Acinetobacter baumannii TOF

Chemical synthesis (α-D-GlcpNAc-(1→2)-[ α-D-ManpNAc-(1→3)-] α-L-

Rhap-(1→2)-α -L-Rhap-(1→3)-α -L-Rhap)

(Zhang, et al., 2008c)

Phosphatidylinositol mannosides (PIMs) from

Mycobacterium tuberculosisTOF

Synthesis of all PIMs including phosphatidylinositol (PI) and

phosphatidylinositol mono- to hexa-mannosides (PIM1 to PIM6)

(Boonyarattanakalin, et al., 2008)

Spore-surface pentasaccharide from Bacillus anthracis Solid-phase synthesis MALDI (Werz, et al.,

2007a) Sulfolipid 1 from

Mycobacterium tuberculosis MALDI Trehalose 2-sulfate with four acyl substituents

(Leigh & Bertozzi, 2008)

Trisaccharide repeat from Xanthomonas campestris pv.

Campestris 8004

→3)-[α -D-Fucp3NAc-(1→2)-β -D-Rhap-(1→3)-α -D-Rhap-(1→

(Comegna, et al., 2008)

Tetra-acylated lipid A derivatives from

Porphyromonas gingivalisTOF Found to be potent antagonists of human

TLR4(Zhang, et al.,

2008e)

Tetrasaccharide - fluorescent label from K30 Antigen

TOF(CHCA)

TOF(DHB)

Dansyl label added with click chemistry (Cheng, et al., 2007)

Tetrasaccharide phosphate from Mycobacterium tuberculosis

cell wall TOF Chemical synthesis from monosaccharide

building blocks (Lee, et al., 2008d)

Carbohydrates from bacteria

α

& HARVEY



S-layer glycoprotein from Geobacillus stearo-

thermophilus NRS 2004/3a

TOF/TOF (DHB)

Construction of self-assembly S-layer glycoproteins as nanopatterned

biomaterials

(Steiner, et al., 2008b)

Nematode etc. carbohydrates

Schistosoma glycans TOF Complex type from eggs. Chemical synthesis

(Nakano, et al., 2007)

Synthetic glycosides α-Amino acid esters with α-D-

Manf side chain Synthesis and antiviral evaluation TOF (DHB) (Ali & Abdel-Rahman, 2008)

Aminoglycosides TOF(CHCA)

Synthesis of pseudodisaccharide, basicity and antibacterial activity

(Chen, et al., 2008c)

TOF (DHB) Arbutin glucosides Use of glucansucrase from Leuconostoc mesenteroides

(Moon, et al., 2007b)

Aryl-β -D-galactopyranosides R-TOF(DHB) Use of phase-transfer catalyst (Kröger & Thiem,

2007)

Biantennary glycan plus phospholipid

TOF(THAP, DHB)

To obtain antibody to N-glycan (Murakami, et al., 2008)

4-(4’-Butoxyphenyl)phenyl-β -O-D-glucoside TOF Synthesized as a low-molecular-mass

gelator (Cui, et al., 2008a)

TOF Lactose hexanoate For preparation of resin to remove anti-α- Gal antibodies from human serum (Jang, et al., 2008)

C-Galactosides TOF (DHB CHCA)

Non-hydrolysable C-galactosides as cholera toxin inhibitors

(Podlipnik, et al., 2007)

Coumarin-conjugated glycosides For monitoring glycosidase activity TOF (Park & Shin,

2007)

Galactosylated cyclooctenone R-TOF(DHB)

Synthesis from lactose. Intermediate for natural product synthesis

(Jürs & Thiem, 2007)

2-C-Glycosylated benzimidazoles TOF (DHB) As antifungal agents but not active

against five pathogenic yeasts (Vojtech, et al.,

2007) 4-Hydroxyphenyl-glucoside

dimers Peroxide-catalysed polymerization TOF (Kiso, et al., 2007)

Ketopyranosyl glycosides R-TOF(THAP)

Anomeric configuration from carbon-proton couplings

(Májer, et al., 2007)

β-1,4-di- D-mannuronic acid glycosides MALDI Glycosides with diacylglycerol as toll-

like receptor ligands (Jiang, et al., 2007)

MALDI OSW Saponins From Ornithogalum Saudersiae with modified sugars (Tang, et al., 2007)

Use of sonication to speed reaction MALDI Oxazolidinone conjugates (Zhang, et al., 2007d)

Phenol glycosides and related compounds from GlcA

TOF(THAP) Synthesis of potential drug metabolites (Arewång, et al.,

2007)

Propyl di-glycosides LTQ (DHB,

Acidic fullerene)

To study fragmentation revealing linkage position and residue identity

(Zhang, et al., 2008b)

Quercetin-α -D-glucopyranosides TOF (DHB) Synthesis with glucansucrase from

Leuconostoc mesenteroides(Moon, et al.,

2007a)

α-Selenoglycosides MALDI Alkyl and aryl, selenoglycosyl amino acids, selenodisaccharides

(Nanami, et al., 2007)

TOF Sugar-cholesterol conjugates GlcNAc, Man and Gal linked with PEG as gene delivery system

(Masuda, et al., 2008)

Tris-phosphorylated ester of Me β-D-Xylp TOF By reaction with 5,5-dimethyl-2-chloro-

1,3,2-dioxaphosphorinane (Pugashova, et al.,

2008)




Bivalent and monovalent arabinofuranoside-containing

alkyl glycosides TOF Synthesis and activity against

mycobacterial growth (Naresh, et al.,

2008)

Fatty acid modified chitobiose TOF (DHB) Lipase-catalysed condensation of

chitobiose and myristic acid in low-water acetone media

(Kuroiwa, et al., 2007)

Fluorescent glycosphingolipids TOF (DHB) With gala oligosaccharides. Use of novel endoglycoceramidase

(Ishibashi, et al., 2007a)

Neoglycolipids TOF(HABA)

Synth. by oxime ligation for microarray analysis of protein interactions (Liu, et al., 2007c)

Neoglycolipids TOF Man3 + dipalmitoylphosphatidyl-ethanolamine for binding studies

(Sakai, et al., 2007a)

Glycosylarchaeols MALDI Synthetic archaeal lipid adjuvants (Sprott, et al., 2008)

Mannosyl glycolipids MALDI From polar lipids extracted from Halobacterium salinarum

(Whitfield, et al., 2008)

β-(1→4)-oligo-D-mannuronic acid neoglycolipids L-TOF, ESI With neo-pentylglycol and two hexadecyl

chains (Xu & Jiang, 2008)

Phosphatidyl-β -D-glucoside TOF, ESI, NMR

To probe antigen selectivity of monoclonal antibody ‘DIM21’

(Greimel, et al., 2008)

1,3-Di-O-tetradecyl-2-O-[4’-O-α-D-Glcp)-β-D-Glcp]-sn-

glycerol TOF To determine biophysical properties (Garidel, et al.,

2008)

CyclodextrinsAlkyl derivatives (regioselective)

R-TOF(CHCA) With vinyl acetate or laurate (Yu, et al., 2007a)

Aminocyclodextrin carboxylic acids

MALDI, ESI

Selective amination and oxidation of 6A,6D-dihydroxy α- and β-CDs

(Hanessian, et al., 2008)

Anthracene-appended α-cyclodextrin MALDI Photocyclodimerization mediated by

cyclodextrin and cucurbit[8]uril (Yang, et al.,

2008a)

Azobenzene-substituted β-CD TOF Single azobenzene group on β-face (Casas-Solvas &

Vargas-Berenguel, 2008)

Azobenzene-substituted β-CD TOF

(CHCA, DHB

Single azobenzene group on β-faceprepared by two methods

(Casas-Solvas, et al., 2008)

Bis-β -cyclodextrin pseudo-cryptand

MALDI (CHCA)

Two β-CDs with urea linkage to chiral diaza-crown ether. Complexes with

busulfan antercancer agent

(Menuel, et al., 2007)

Bis-β -cyclodextrin linked with ethylene oxide

TOF (DHB), ESI Complexes with adamantane (Bistri, et al., 2007)

Bis- β-cyclodextrin linked with stilbene TOF (DHB) For construction of supramolecular

structures (Kuad, et al., 2007)

β-Cyclodextrin dimers TOF/TOF (DHB) Synthesis by click chemistry (Mourer, et al.,

2008)

β-CD fatty acid esters MALDI Chain lengths (C4---C14) synthesised by

trans-esterification of β-CD by vinyl fatty ester using thermolysin in DMSO

(Choisnard, et al., 2007)

β-CD linked to azobenzene viaPEG linker TOF (DHB) For studies on thermal and photochemical

conformation switching (Inoue, et al.,

2007b) β-CD linked to Ru(II)-

terpyridine TOF Encapsulation by CD. Article in Korean (Park, et al., 2007a)

β-CD monoesterified with 3- Forms large aggregates wTOF (Silva, et al., 2008) ith two

Glycolipids

((E)-dec-2-enyl)-dihydrofuran-2,5-dione recognition sites



β-Cyclodextrin substituted by 2-pyridyl triazole TOF Generation of new fluorophore by click

chemistry (David, et al.,

2007) Bis(pyrene)-functionalized, per-

Bz β-CD TOF

(dithranol) Gives blue or green fluorescence

depending on solvent (Huo, et al., 2008)

CDs with guanidinoalkylamino and aminoalkylamino groups on

their primary side Synthesis, characterization and properties TOF (DHB) (Mourtzis, et al.,

2008)

5-N-β -Cyclodextrin-carboxamide-5-methyl-1-

pyrroline N-oxide MALDI

Improved spin trapping. βCD can improve adduct stability via intra-

molecular interaction with O2 •- adduct (Han, et al., 2008)

TOF (DHB) Cyclodextrin methacrylate Via microwave-assisted click reaction (Munteanu, et al., 2008)

Dansylcysteine and tosyl substituted γ-CDs

R-TOF(DHB), PSD

(CHCA) (Yu, et al., 2007c) Substituted groups at adjacent positions

Dimaltosyl-β -cyclodextrin TOF (DHB) Transglycosylation reaction using TreX, a

Sulfolobus solfataricus P2 debranching enzyme

(Kang, et al., 2008b)

Diphisphane-CD (TRANSDIP)

TOF(CHCA,

dithranol) ESI

Synthesis of an authentic trans-spanning ligand

(Poorters, et al., 2007)

Di(thio-1,2-dicyane ethylenylthio)ethane-tethered β-

cyclodextrin dimer TOF

Shows unusual electrochemical effect upon complexation with ferrocene in

DMF solution (Lu & Lu, 2007)

Di- and tetra-(6-deoxy-6-alkylthio)- and 6-

(perfluoroalkypropanethio)-α -CD derivatives

MALDI Synthesis of twelve molecules with groups on the primary face

(Bertino-Ghera, et al., 2008)

6I,6n-Di-O-(L-Fucp)-β -CD (n=II-IV)

TOF (DHB), FAB (Nishi, et al., 2007) Synthesis and haemolytic activity

Gd-containing complex plus β-CD MALDI Synth. of multimeric magnetic resonance

contrast agents for cellular imaging (Song, et al., 2008)

D-Galactose-β -cyclodextrin conjugates (Oda, et al., 2008) As drug-carrying molecules TOF

Inclusion complexes with guaiacol TOF (DHB) 1:1 Solid complexes with β- and γ-CDs (Song, et al., 2007)

Milk sugar-CD conjugates TOF (DHB

+ve, THAP -ve)

Synthesis by reductive amination. CDs oxidized at C6 with TEMPO

(Weikkolainen, et al., 2007b)

(Weikkolainen, et al., 2007a)

Mono-(6-O-(p-tolylsulfonyl))- β-cyclodextrin TOF To construct carbon nanotubes with

covalently-bound CDs (Klink & Ritter,

2008)

N-Glycosyl-thiocarbamoyl-CD TOF (DHB)

α-D-Man and β-L-Fuc residues with hexyl-, dodecyl- and hexadecyl thio

chains at 6-position and glycosylthiocarbamoyl-oligo-ethylene

glycol chains at 2-position

(McNicholas, et al., 2007)

Phosphate-substituted α- and β-cyclodextrins

TOF(CHCA)

With one or two phosphates on primary rim

(López & Bols, 2008)

Polyethyleneglycol substituted β-CD

R-TOF(DHB) For study of self-threading dynamics (Inoue, et al.,

2007a)




Polyester substituted α-, β-, γ-CD TOF

CDs found to initiate ring-opening polymerizations of lactones selectively to

give polyesters in high yields

(Osaki, et al., 2007a)

Polystyrene linked to β -CD (Felici, et al., 2008) Aggregates into vesicles TOF

TOF Polysulfonated CDs Synthesis of five regioisomeric β-cyclodextrin tetramesitylenesulfonates

(Yamamura, et al., 2007)

Propylene-bridged α -CD TOF(CHCA)

For study of catalytic properties in oxidation reactions

(Lopez, et al., 2007)

Pseudo[1]rotaxane TOF(CHCA) From a flexible cyclodextrin derivative (Miyawaki, et al.,

2008b)

CDs threaded onto a polymer chain TOF Pseudo-rotaxane-CDs (Osaki, et al., 2007b)

TOF (DHB) Pyrene-CD adduct For synthesis of CD-carbon nanotube hybrids

(Ogoshi, et al., 2007)

[2]Rotaxanes bearing α-cyclodextrin derivatives TOF

To investigate relative rotary movement of a ring relative to an axis molecule and

that of an axis molecule in a ring

(Nishimura, et al., 2008)

TOF (DHB) Rotaxanes Di-Me-β-cyclodextrin and oligothiophenes. Suzuki coupling

(Sakamoto, et al., 2007)

Sulfated cyclodextrins TOF (Six matrices)

Degree of sulfation by MALDI. As antimalarials

(Crandall, et al., 2007)

6,6’-telluroxy-bis(6-deoxy-β -cyclodextrin) and related

compounds MALDI Demonstration that telloroxides exhibit

hydrolysis capacity (Dong, et al., 2007)

Thiol-modified β-CD TOF Construction of gold nanoparticles for fingerprint detection

(Becue, et al., 2007)

Cyclodextrin plus γ-valerolactone TOF Ring-opening polymerization of cyclic

esters by cyclodextrins (Harada, et al.,

2008) Polymers

Amphiphilic block copolymers TOF Self-association of bis-(α,β-D-glucopyranosyl)-polyisobutylene

(Orosz, et al., 2007)

Biotin α-end-functionalized glycopolymers

TOF(CHCA)

RAFT copolymerization of acrylamide derivs. of Gal with N-acryloylmorpholine

in the presence of a biotin CTA(Gody, et al., 2008)

Branched supramolecular polymers from CDs TOF

From 3-cinnamamide- α-CD and 3-N -

cinnamamidehexane-carbonyl-Nε -cinnamamide-lysinamide- α-CD

(Miyawaki, et al., 2008a)

Cellulose acetate modified with caprolactone R-TOF Various reaction conditions. Caprolactone

chains mainly at positions 2 and 6(Számel, et al.,

2008) Dextran-coated styrene

nanoparticles R-, L-TOF

(DHB) Emulsion-polymerization of styrene in

the presence of dextran (Ladaviere, et al.,

2007)

1,2:3,4-di-O-isopropylidene-6-O-(2′- formyl-4′-vinylphenyl)-

D-Galp (IVDG) polymer

TOF(CHCA)

Polymerization of IVDG achieved with 2,2'-azobis(isobutyronitrile) as initiator

and 1-PhEt dithiobenzoate as RAFT agent at 60°C

(Xiao, et al., 2008)

Galactose-functionalized poly-L-glutamic acid-based

glycopolymers

TOF(sinapinic)

To study effect of saccharide spacing on multivalent binding

(Polizzotti, et al., 2007)

TOF (DHB) Lactose-substituted polystyrene By nitroxide-mediated radical

polymerization of styrene carrying acetylated lactose

(Miura, et al., 2007a)

Methacrylamide-carbohydrate polymers (Tsuji, et al., 2007) Methacrylamides with bulky side groups MALDI

α

& HARVEY



MeOH, dioxane or vancomycin template ESI gave chains) 2008)

Polylactide L-TOF (dithranol)

Polymerization co-initiated by carbohydrate esters and pyranoses (Tang, et al., 2008)

TOF Sialic acid-containing polymers For use in attenuation of β-amyloid peptide in Alzheimer’s disease

(Cowan, et al., 2008)

Vinyl polymers with laterally attached 4,4′′-digalactosyloxy-

p-terphenyl side groups Synthesis and chiroptical properties TOF (Cui, et al., 2008b)

Antibiotics

Aminocoumarin antibiotics TOF(sinapinic)

Chemoenzymatic formation of novel aminocoumarin antibiotics by the

enzymes CouN1 and CouN7

(Fridman, et al., 2007)

Chloramphenicol-neomycin conjugate TOF Bacteriophage links to neomycin to carry

chloramphenicol to target (Yacoby, et al.,

2007)

TOF Doxorubicin prodrug Drug activated by Staudinger reaction (triphenylphosphine not toxic as used)

(van Brakel, et al., 2008)

1,1’-Linked α-L-lyxopyranosyl β-D-glucopyranoside

TOF(CHCA)

Intermediate in the biosynthesis of avilamycin

(Schmidt & Wittmann, 2008)

Microarrays

Glycopeptide microarray R-TOF(THAP)

Enzymatic attachment of GalNAc to peptide array on gold surface

(Laurent, et al., 2008c)

Mannose-decorated thioacetates TOF (nor-harmane)

For construction of self-assembled glycan arrays utilizing 1,3-dipolar cycloaddition

(Kleinert, et al., 2008)

MALDI Paromomycin derivatives Modified at C5’’. Less active than parent compound

(Kudyba, et al., 2007)

TDP-L MALDI -digitoxose For investigations into the biosynthesis of Kijanimicin

(Zhang, et al., 2007b)

Trisaccharide epitope of extracellular proteoglycan from

Microconia prolifera

R-TOF(DHB)

On gold nanoparticles to study species-specific cell adhesion

(Carvalho de Souza, et al., 2008)

TOF (DHB) Xyloglucans Glyco-array for monitoring plant cell wall glycosyltransferase activities

(Shipp, et al., 2008)

Miscellaneous

TOF (DHB) 7-Acetoxy-4-methylcoumarin Methanolysis catalysed by cyclosophorases from Rhizobium meliloti (Park, et al., 2008a)

Acrylic acid esters of mono- and di-saccharides TOF Esterification of free carbohydrates from

sugar cane (Tsukamoto, et al.,

2008) N-Alkylated 1-

deoxymannojirimycin derivatives

R-TOFFluorescent iminoalditol-amino acid hybrids with glucosidase inhibitory

properties.

(Steiner, et al., 2007)

Amphiphilic cyclic disaccharide analogues TOF Two phosphodiester links, four lipophilic

chains (Coppola, et al.,

2007)

TOF Biotinylated glyconanoparticles Reduction with poly(ethyleneglycol), and

HAuCl4 via a photochemical process gives biotinylated gold nanoparticles.

(Miyagawa, et al., 2007)

bis-(α , β-D-glucopyranosyl)-polyisobutylene To study self-association TOF (Orosz, et al.,

2007) 1-Deoxygalactonojirimycin-

lysine hybrids As potent TOF D-galactosidase inhibitors (Steiner, et al., 2008a)

Fused bicyclic thioglycosides of GlcNAc As mycothiol biosynthesis inhibitors TOF (Slättegård, et al.,

2007)

Galactose-cyclohexenephosphonates

TOF (3,5-DHB, ATT,

To find competitive inhibitors of the trans-sialidase of the pathogen (Busse, et al.,

2007)

Poly 1,6-dithio-DEffect of solvent (HTOF (DHB), -mannitol

2 (Bereczki, et al., O, DMF gave rings,

CHCA) Trypanosoma cruzi


HQ hydroxyquinoline (8-hydroxyquinoline)HSA human serum albuminHUPO Human Proteome OrganizationHSQC heteronuclear single quantum coherenceIAA 3-indoleacrylic acidICR ion cyclotron resonanceIdo idoseIEF isoelectric focusingIgA (G) immunoglobulin A (G)Ile iso-leucineImCHCA 1-ethylimidazolium-a-cyano-4-hydroxycinna-

mateIMS ion mobility spectrometryIR infraredIRMPD infrared multiphoton dissociationIT ion trapIUPAC International Union of Pure and Applied

ChemistryKEGG Kyoto Encyclopedia of Genes and GenomesL- linear (as in linear-TOF)Lac lactoseLDI laser desorption ionizationLC liquid chromatographyLH luteinizing hormoneLOS lipooligosaccharideLPS lipopolysaccharides

L-SIGN liver/lymph node specific intercellular adhesionmolecule-3-grabbing non-integrin

LTQ linear ion trapLys lysineMALDI matrix-assisted laser desorption/ionization mass

spectrometryMan mannoseManNAc N-acetylmannosaminemf-MELDI matrix-free material-enhanced laser desorption/

ionizationMMT 3-methyl-1-p-tolyltriazineMNP magnetic nanoparticlesMS mass spectrometrym/z mass to charge ratioMUC mucinNBD nitrobenz-2-oxa-1,3-diazoleNBDNJ N-butyl-deoxynorimycinNeu5Ac N-acetylneuraminic (sialic) acidNeu5Gc N-glycoylneuraminic acidNMR nuclear magnetic resonanceNOESY nuclear overhauser enhancement spectroscopyNP normal phaseOR optical rotationORD optical rotary dispersionOS oligosaccharidep (as in Glcp) pyranose form of sugar ring


Glycoconjugated chlorin p6cycloimide MALDI

Simultaneous glycosylation (D-Gal) of macrocycle and replacement of oxygen in additional ring of purpurin 18 by nitrogen

(Lebedeva, et al., 2007)

Synthesis by cross-metathesis reaction TOF Glycoporphyrins (da Silva, et al., 2008)

TOF Glyco nanoribbons Carbohydrate, PEG spacer and β-sheet-forming peptide for self assembly (Lim, et al., 2007b)

Lactose-conjugated psoralen TOF(CHCA)

For preparation of spherical DNA micro-assemblies (Kim, et al., 2007e)

N4- β-D-glycoside cytosines (Ali, et al., 2007) As antiviral agents TOF (DHB)

Neu5Ac-Gal-GlcNAc plus nitrophenol and biphenyl

FTMS(DHB)

Bifunctional CD22 ligands use multimeric immunoglobulins as protein

scaffolds in assembly of immune complexes on B cells

(O’Reilly, et al., 2008)

O-Oligosaccharidyl-1-amino-1-deoxyalditols TOF (DHB) Reducing sugar + NH4HCO3 +

NaCNBH3. Dimers with high sugar conc. (Miller, et al.,

2007) Porphyrin (chlorin)

glycoconjugates TOF/TOF

(DHB) Click chemistry to obtain photosensitizers

for cancer chemotherapy (Grin, et al., 2008)

TOF (DHB) Salacinol analogues With carboxylate inner salt as human maltase glucoamylase inhibitor (Chen, et al., 2007)

TOF (DHB) Salacinol analogues Phosphate analogues as glycosidase inhibitors (Bhat, et al., 2007)

(Nasi, et al., 2007) Chain modified as glycosidase inhibitors TOF (DHB) Salacinol and blintol analogues 3,4,5-Tri-O-acetyl-2-

[18F]fluoro-2-deoxy-D-glucopyranosyl 1-

phenylthiosulfonate

TOF Thiol-reactive agent for the

chemoselective 18F-glycosylation of peptides

(Prante, et al., 2007)

1H-(1,2,3-triazol-1-yl)-mannosides As selective galectin-3 and 9N inhibitors TOF (Tejler, et al.,

2007) 1Format (not all items present): MALDI method (matrix), derivative. ‘‘MALDI’’ is used when the instrument is not specified.

& HARVEY


TABLE 22. Use of MALDI MS to study methods for general synthesis

Compound Method1 ecnerefeRsetoNAcylated

carbohydrates TOF Indium(III) triflate used as catalyst for acyl transfer reactions (Giri, et al., 2008)

Aryl glycosides TOF/TOF Use of planetary ball mill to achieve solvent free synthesis

(Patil & Kartha, 2008b)

Biotin derivatives R-TOF (DHB) As multifunctional oligosaccharide tags (Chindarkar &

Franz, 2008)

Carbohydrate units (general)

TOF(CHCA),

ESI

Preparation and use of p-alkoxyphenyl-type heavy fluorous tag

(Mizuno., et al., 2008)

Cello- and chito-oligomers TOF Use of galactosyltransferase and α-lactalbumin to

transfer galactose to glucose (Streicher, et al.,

2008) 1,2-Cis-

glycosylation TOF (DHB),

ESI Synthesis by naphthylmethyl ether mediated

intramolecular aglycon delivery (Ishiwata, et al.,

2008b)

Derivatized hexosides R-TOF

Regioselective 6-O-tritylation/silylation of various monosaccharides or their derivatives under solvent-free

conditions by dry grinding

(Patil & Kartha, 2008a)

Disaccharides (1→6-linked) TOF Microwave-assisted stereospecific intra-molecular

rearrangement catalyzed by Mo(VI)(Hricovíniová,

2008) Glc-β-(1→6)-Man-α-(1→6)-

Glc-β-1→pentenyl trisaccharide

MALDI

“Cap-and-tag” strategy for solid phase oligosaccharide synthesis. Ac-capping and fluorous-tagging allowed

separation of F-tagged oligosaccharide from Ac capped deletion sequences using fluorous extraction.

(Carrel & Seeberger, 2008)

Glycans with β-mannose linkages MALDI First automated solid-phase method for synthesis

containing difficult β-Man linkages (Codée, et al.,

2008)

Glycopeptides TOF (DHB) Use of sugar-assisted ligation with complex oligo- rather than with mono-saccharides

(Bennett, et al., 2008)

Glycopeptides TOF Cysteine-free peptide and glycopeptide ligation by direct aminolysis

(Payne, et al., 2008)

Glycoproteins R-TOF (DHB)

6-Azido D-Gal transfer to protein with terminal GlcNAc using commercially available β-1,4-

galactosyltransferase

(Bosco, et al., 2008)

Glycoproteins L-TOF (sinapinic)

Synthesised by improved method involving reductive amination

(Gildersleeve, et al., 2008)

Glycoproteins TOF, ESI Use of novel linker method for preparation of conjugate vaccines

(Dziadek, et al., 2008)

Glycosyl azides TOF/TOF Solvent-free synthesis from glycosyl halides by ball milling

(Mugunthan & Kartha, 2008)

Lewisx derivatives TOF (DHB) Use of microwave effects at low temperature (Shimizu, et al., 2008)

Malto-oligosaccharides TOF (DHB) Enzymatic α-glucosaminylation catalyzed by

phosphorylase (Nawaji, et al.,

2008a)

Monosaccharides TOF Use of fluorous tag (-Ph-O-C37O3F51) and fluorous-organic partition

(Goto & Mizuno, 2007)

N-Protected glycosylamines TOF One-step synthesis from sugar hemiacetals (Liautard, et al.,

2008)Neoglyco-conjugates TOF Peptide-sugar ligation catalyzed by transpeptidase

sortase(Samantaray, et al.,

2008) Neoglycolipids GlcNAc, Gal-

GlcNAc TOF Study of the efficiency of cyclodextrins for

neoglycolipid synthesis using glycosyltransferases (Nagashima, et al.,

2008)



PAA porous anodic aluminaPAD pulsed amperometric detectionPAGE polyacrylamide gel electrophoresisPAMAM poly(amidoamine)PBH pyrenebutyric acid hydrazidePECAM platelet endothelial cell adhesion moleculePEG polyethylene glycol

Pen pentosePhe phenylalaninePI phosphatidylinositolPIM phosphatidyl-myo-inositol mannosidePMP 1-phenyl-3-methyl-5-pyrazolonePNGase protein-N-glycosidasePSA prostate-specific antigen


Oligosaccharides MALDI Combination of reductive openings of benzylidene acetals and glycosylations

(Vohra, et al., 2008)

Oligosaccharides TOF Use of glycosyl phthalates as donors (Kim, et al., 2008d)

Oligosaccharides TOF Use of 4,6-dimethoxy-1,3,5-triazin-2-yl-lactoside and donor with endo-1,4-β-glucanase

(Tanaka, et al., 2008b)

Oligosaccharides Trap-TOF (DHB)

Synergistic solvent effect in 1,2-cis-glycoside formation

(Ishiwata, et al., 2008a)

Oligosaccharides TOF Glycosylation involving gold colloidal nanoparticles. Reagent remnants could be removed by ultrafiltration

ready for MALDI

(Shimizu, et al., 2007)

Oligosaccharides TOF By TiCl4-promoted ring opening of cyclodextrin derivatives

(Bösch & Mischnick, 2007)

Oligosaccharides, glycolipids MALDI Gold(I)-catalyzed glycosylation of 1,2-anhydrosugars

gives increased yields (Li, et al., 2008h)

Proteoglycan core structures

TOF/TOF(DHB) Synthesis from 1,6-anhydro-β-lactose (Shimawaki, et al.,

2007)

Sialylatedoligosaccharides

TOF(CHCA)

Sialylation reactions with 5-N,7-O-carbonyl-protected sialyl donors show β-selectivity with nitrile solvent

(MeCN) assistance

(Tanaka, et al., 2008a)

Sugar alkynols TOF Investigation of competitive 5-exo-dig versus 6-endo-dig cyclizations

(Ramana, et al., 2008)

Triazole-linked C-disaccharide TOF Validation of the copper(I)-catalyzed azide-alkyne

coupling in ionic liquids (Marra, et al.,

2008a)

Various TOF, ESI Use of whole cellular preparations with a saccharide primer to produce libraries of oligosaccharides (Sato, 2008)

Various TOF (DHB), PSD Preparation of saccharide primers for above method (Sato, et al., 2008)


TABLE 23. Use of MALDI MS to study carbohydrate reactions

sdohteMnoitcaeR 1 Reference Acetolysis of 6-deoxy-sugar oligosaccharide building blocks. Exo

versus endo activation TOF (Cirillo, et al., 2008)

Benzoate methanolysis catalyzed by α-cyclosophorohexadecaose isolated from Xanthomonas oryzae TOF (DHB) (Cho, et al., 2007)

Bradyrhizobial cyclic β-(1→3),(1→6)-glucans as chiral additives for enantiomeric separation of flavanones, by CE TOF (DHB) (Kwon, et al.,

2007a) Cyclosophorohexadecaose and succinoglycan monomers as

catalytic carbohydrates for the Strecker reaction TOF (DHB) (Lee, et al., 2007f)

Hydrolysis of digoxin catalysed by cyclodextrin dicyanohydrins TOF (Bjerre, et al., 2008)

Selective acetolysis of 6-deoxy-sugar oligosaccharide building blocks MALDI (Bedini, et al.,

2008) Selective cleavage of sugar anomeric O-acyl groups using

FeCl3.6H2OTOF (Wei, et al., 2008b)


& HARVEY


PSD post-source decayPsi psicosePTFE polytetrafluoroethylenePVDF polyvinylidine difluorideQ quadrupoleQIT quadrupole ion trapQ quadrupoleQIT quadrupole ion trapQui quinovose (6-deoxyglucose)R- reflectron (as in R-TOF)RAFT reversible addition-fragmentation chain

transferRha rhamnose (6-deoxymannose)rHuEPO recombinant human erythropoietinRib riboseRNase ribonucleaseRP reversed phaseRSD relative standard deviationSALDI surface-assisted laser desorption/ionizationsDHB super-DHBSDS sodium dodecyl sulfateSEC size-exclusion chromatographySer serineSMALDI scanning microprobe matrix-assisted laser

desorption/ionizationSNA Sambucus nigra agglutinin-T(as GlcNAc-T) transferaseTag tagatoseTFA trifluoroacetic acidTFMS trifluoromethanesulfonic acidTHAP trihydroxyacetophenoneTIMP tissue inhibitor of metaloproteinaseTLC thin-layer chromatographyTMG 1,1,3,3-tetramethylguanidiniumTMS trimethylsilylThr threonineTLC thin-layer chromatographyTOF Time-of-flightTPPOC3Py 5,10,15,20-tetrakis[4-(3-pyridiniumpro-

poxy)phenyl]prophyrin tetrakisbromideUDP uridine diphosph(ate)(o)UPLC ultra performance liquid chromatographyUV ultravioletUVPD ultraviolet photodissociationWGA wheat germ agglutininXyl xyloseYAG Yttrium aluminium garnet

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