1

Comparison of Methods for Profiling O-glycosylation: HUPO

Human Disease Glycomics/Proteome Initiative Multi-

Institutional Study of IgA1

Yoshinao Wada1,2,4, Anne Dell1,3,5, Stuart M. Haslam3,5, Bérangère Tissot3,5, Kévin

Canis3,5, Parastoo Azadi6, Malin Bäckström7, Catherine E. Costello8, Gunnar C.

Hansson7, Yoshiyuki Hiki9, Mayumi Ishihara6, Hiromi Ito10, Kazuaki Kakehi11,

Niclas Karlsson12, Koichi Kato13,14, Nana Kawasaki15, Kay-Hooi Khoo16, Kunihiko

Kobayashi17, Daniel Kolarich18, Akihiro Kondo19, Carlito Lebrilla20, Miyako

Nakano18, Hisashi Narimatsu10, Jan Novak21, Milos V. Novotny22, Erina Ohno14,

Nicolle H. Packer18, Matthew B. Renfrow21, Michiko Tajiri4,23, Kristina A.

Thomsson7, Hirokazu Yagi14, Shin-Yi Yu16, and Naoyuki Taniguchi2,24,25,26

4Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi,

Japan

5Division of Molecular Biosciences, Department of Life Sciences, Imperial College

London, London, UK

6Analytical Services, Complex Carbohydrate Research Center, University of Georgia,

Athens, GA, USA

7Dept. Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden

8Mass Spectrometry Resource, Department of Biochemistry, Boston University

School of Medicine, Boston, MA, USA

9Department of Internal Medicine, Fujita Health University School of Medicine,

Toyoake, Japan

MCP Papers in Press. Published on January 4, 2010 as Manuscript M900450-MCP200

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

2

10Research Center for Medical Glycoscience, National Institute of Advanced

Industrial Science and Technology, Tsukuba, Japan

11Faculty of Pharmaceutical Sciences, Kinki University, Higashiosaka, Japan

12Chemistry Department, National University Ireland, Galway, Ireland

13Institute for Molecular Science and Okazaki Institute for Integrative Bioscience,

National Institutes of National Sciences, Okazaki, Japan

14Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya,

Japan

15Division of Biological Chemistry and Biologicals, National Institute of Health

Sciences, Tokyo, Japan

16NRPGM Core Facilities for Proteomics and Glycomics, Institute of Biological

Chemistry, Academia Sinica, Taipei, Taiwan

17Department of Pediatrics, Hokkaido University Graduate School of Medicine,

Sapporo, Japan

18Department of Chemistry and Biomolecular Sciences, Macquarie University, Sidney,

Australia

19Laboratory of Molecular Diagnostics and Informatics, Division of Health Sciences,

Osaka University Graduate School of Medicine, Suita, Japan

20Department of Chemistry, University of California Davis, Davis, CA, USA

21Department of Microbiology and Biochemistry and Molecular Genetics, University

of Alabama at Birmingham, Birmingham, AL, USA

22Department of Chemistry, Indiana University, Bloomington, IN, USA

23CREST, Japan Science and Technology Agency, Kawaguchi, Japan

24Department of Biochemistry, Osaka University Graduate School of Medicine, Suita,

Japan

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25 The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan

26Systems Glycobiology Group, Disease Glycomics Team, RIKEN Advanced Science

Institute, Wako, Japan

1To whom correspondence should be addressed: e-mail waday@mch.pref.osaka.jp;

a.dell@imperial.ac.uk

2 Study co-ordinators

3 Study assessors

4

ABSTRACT

The Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome

Initiative (HGPI) recently coordinated a multi-institutional study which evaluated

methodologies that are widely used for defining the N-glycan content in glycoproteins.

The study convincingly endorsed mass spectrometry as the technique of choice for

glycomic profiling in the discovery phase of diagnostic research. The present paper

reports the extension of HGPI’s activities to an assessment of the methodologies

currently employed for O-glycan analysis. Three samples of IgA1 isolated from the

serum of patients with multiple myeloma were distributed to fifteen laboratories

worldwide for O-glycomic analysis. A variety of mass spectrometric and

chromatographic procedures were employed, representative of current methodologies.

Similar to the previous N-glycan study, the results convincingly confirmed the pre-

eminent performance of mass spectrometry (MS) for O-glycan profiling. Two

general strategies were found to give the most reliable data, namely direct MS

analysis of mixtures of permethylated reduced glycans in the positive ion mode, and

analysis of native reduced glycans in the negative ion mode using LC-MS approaches.

In addition mass spectrometric methodologies to analyze O-glycopeptides were also

successful.

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INTRODUCTION

Recently, the Human Proteome Organisation (HUPO) Human Disease

Glycomics/Proteome Initiative (HGPI), coordinated an evaluation of methodologies

that are widely used for defining the N-glycan content in glycoproteins (1). Twenty

laboratories around the world participated in the study in which the glycosylation of

standard samples of transferrin and immunoglobulin-G (IgG) was characterized by a

variety of chromatographic and mass spectrometric techniques. Two clear messages

emerged from this study. Firstly, there was significant variance amongst the datasets

from laboratories employing chromatographic profiling which was likely due to

incomplete derivatization with the fluorophores that had been employed to “tag” the

oligosaccharides in order to facilitate chromatography and provide a means of

detection. Secondly, mass spectrometry (MS) was shown to give consistent data in

inter-laboratory comparisons and it was concluded that MS-based strategies provide

the most effective means of both identification and quantitation of N-glycans in

glycomic studies.

HGPI has now extended its comparison of analytical methodologies to

encompass mucin-type O-glycosylation. In this type of glycosylation, O-glycans are

attached by a GalNAc to the amino acids serine and threonine. They can occur as

single O-glycans or clustered in mucin domains. Such domains are most abundant in

the class of glycoproteins known as mucins, which typically have a great number of

mucin domains arranged as tandem repeats, but are also found in many extracellular

proteins like IgA, which is the subject of the current study. IgA1 represents one of

two structurally and functionally distinct subclasses of IgA (2). The heavy chains of

IgA1 molecules contain a hinge-region segment between the first and second constant

region domains. This segment, which has a high content of Pro, Ser, and Thr, is the

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site of attachment of usually up to six O-linked glycan chains (3-9). In circulatory

IgA1, these O-glycans consist of GalNAc with a ß1,3-linked Gal; both saccharides

may be sialylated (3, 4). The carbohydrate composition of the O-linked glycans in the

hinge-region of normal human serum IgA1 is variable. The prevailing forms include

Gal-GalNAc disaccharide, and its mono- and di-sialylated forms (4, 5, 10). Gal-

deficient variants with terminal GalNAc or sialylated GalNAc are rarely found in the

O-glycans of normal serum IgA1 (5), but are much more common in IgA nephritis

patients (11, 12), in whom IgG autoantibodies reactive to the hypogalactosylated

IgA1 form immune complexes and lead to mesangioproliferative glomerulonephritis

(13). These pathological features clearly demonstrate the importance of determining

the profile of the total glycan pool as an initial but essential step in tackling the

complex O-glycan structures. In the present study, IgA1 preparations from three

patients with multiple myeloma were delivered by HGPI to 15 experienced academic

laboratories and the results of O-glycomic analysis, especially the total glycoform

profiles, obtained using different analytical methodologies, were assessed. This study

was designed to compare and evaluate various methods, differing in sample

preparation and analytical modes, as well as to document levels of variance or

consistency among the data.

EXPERIMENTAL PROCEDURES

Sample Preparation

Three IgA1 myeloma proteins named "NUD", "VDS" and "SapII" were isolated

from respective serum samples from three patients with multiple myeloma by

precipitation with 50% saturated ammonium sulfate, followed by gel filtration

chromatography on Sepharose 6B and by ion exchange chromatography on DEAE

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cellulose (14, 15). Purity was assessed by immunoelectrophoresis with rabbit anti-

human serum proteins. These samples were provided by one of the authors (KK).

Each 250 µg sample was delivered with dry ice in a solution for “NUD” at 250

µg/mL in 6M guanidine/HCl, 0.25M Tris-HCl, 1% dithiothreitol (pH8.6) and for

“Sap-II” at 2 mg/mL in 20mM Tris-HCl, 2% NaCl, 0.1% NaN3 (pH8.0), or a

lyophilized form for “VDS”.

Analytical Protocols

The analytical methods employed by each participating laboratory are given in

the Supplementary Material.

RESULTS AND DISCUSSION

Human IgA1 has both N- and O-glycosylation with the latter being the focus of

this study. The O-glycans are located on the hinge region of the heavy chain and five

sites of glycosylation have previously been identified (Thr-225, Thr-228, Ser-230,

Ser-232 and Thr-236), two of which (225 and 236) have been reported to be partially

occupied (3-6). Tryptic cleavage yields the 38 amino acid hinge-region O-

glycopeptide: HYTNPSQDVTVPCPVPST225PPT228PS230PS232TPPT236PSPSCCHPR.

The mass of core peptide containing carbamidomethylated cysteine residues is

4135.88 (monoisotopic) or 4138.56 (average).

The fifteen laboratories participating in the study analyzed tryptic glycopeptides

or released oligosaccharides, or both. General workflows are shown in Schemes 1

and 2 and methodologies employed by each laboratory are summarized in Table I

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(note that the laboratory numbers designated in Table I are not the same as those

given to the authors in the title). IgA1 derived from three patients with multiple

myeloma (coded NUD, Sap-II and VDS) (14, 15) were prepared in Japan and

dispatched to the participating laboratories in 250 µg aliquots. Several of the

participating laboratories additionally analyzed a sample of IgA pooled from healthy

individuals.

Glycopeptide Analysis

Matrix-assisted laser desorption/ionization (MALDI) and/or electrospray

ionization (ESI) mass fingerprinting of cysteine-alkylated tryptic hinge O-

glycopeptides was carried out by six laboratories. Data of remarkable consistency

were obtained from most of these laboratories despite the employment of a variety of

sample handling procedures and mass spectrometric instrumentation (see Table 1 and

Supplementary Methods). Exemplar MALDI and ESI mass spectra from O-

glycopeptides isolated from solution digests using hydrophilic affinity extraction (16)

by two laboratories (1 and 4) are shown in Figure 1 and Supplementary Figure S1,

respectively. The enriched glycopeptide fraction was directly analyzed by MALDI

MS in linear TOF mode (lab1) or subjected to on-line LC-ESI-MS (lab4). Profiles

obtained from on-line LC-ESI-MS of solution digests and by in-gel digestion

followed by LC-ESI-MS are shown in Figure 2 and Supplementary Figures S2 and S3

(labs 2, 5 and 6, respectively). Some laboratories additionally analyzed a pooled

sample of IgA1 purified from healthy individuals. An exemplar mass fingerprint for

the hinge glycopeptide of normal IgA1 is shown in Supplementary Figure S4. For

simplicity molecular ions of the glycopeptide glycoforms are annotated in all spectra

with their predicted oligosaccharide compositions using N, H and NA to represent

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HexNAc, Hex, and NeuAc, respectively.

Sialylation was prominent in Sap-II, and heterogeneity was small in NUD in all

the data sets. However, some differences were also observed. Typically, sialylated

glycans were less abundant in the MALDI mass spectra (lab1), when looking at the

ratio of 4N4H/4N4H1NA/4N4H2NA/4N4H3NA in Sap-II (Fig. 3) or that of

4N4H1/4N4H1NA in VDS in Figure 1 and Supplementary Figures S1 and S2. This

was likely due to low efficiency of the ionization of sialylated glycopeptides in the

positive ion mode, because the underestimation was less prominent in the MALDI

mass spectra taken in the negative mode (see Supplementary Fig. S5 from lab1). Loss

of sialyl residues is the major drawback of MALDI of oligosaccharides or

glycopeptides, and this was observed, even when using the negative ionization mode,

especially in the glycopeptide ions with multiple sialic acids: e.g., 5N4H3NA and

4N4H4NA (Supplementary Fig. S5). However, the extent of sialic acid loss from O-

glycans is less prominent than the cases of N-glycans (unpublished observations by

YW and MT).

On the other hand, the results from ESI from three laboratories, lab2 (Fig. 2),

lab4 (Supplementary Fig. S1) and lab5 (Supplementary Fig. S2), also showed some

discrepancies in the sialylated species. As indicated by the ratio of 4N4H to

4N4H1NA of NUD, sialylation levels were higher in the analysis of lab4 than in those

of lab 2 and lab5. From the mass spectrometric point of view, one issue that should

be clarified to address the disagreement is the charge-state distribution of the original

mass spectra from these laboratories or the charge-state of the source mass spectra for

deconvolution. Alternatively, the disagreement may be due to the usage of different

types of mass analyzers and detectors. In addition, lab5 reported predominance of

small glycans probably due to different sample preparation (Fig. 3); lab5 extracted

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glycopeptides from SDS-PAGE gels by in-gel digestion.

One laboratory (lab3) employed Jacalin lectin chromatography to isolate the

glycopeptides, but the yields were found to be insufficient for a comprehensive MS

study and the limited data that were obtained differed substantially from the other

laboratories. Considering that this laboratory has sufficient experience of the analysis

of hinge O-glycopeptides obtained from a few mg of IgA1, their protocol was not

suited for a smaller scale analysis; probably the volume (2 mL) of Jacalin was too

much to efficiently recover the glycopeptides from the lectin column.

Additional analyses to decrease the complexity of the MS profiles were carried

out by lab1 (Supplementary Figs. S6 and S7). Desialylation removed the negative

effect of sialic acid on the ionization efficiency in the MALDI, and the small signals

corresponding to highly glycosylated glycopeptides with six or seven HexNAc

residues could be observed (Supplementary Fig. S6). The number of O-glycans

attached to the glycopeptides was clearly demonstrated by the mass spectra after

eliminating peripheral glycans attached to the Ser/Thr-GalNAc units by incubation

with trifluoromethanesulfonic acid (Supplementary Fig. S7). However, the current

protocol of reactions did not guarantee good reproducibility and some removal of

GalNAc residues was observed. Deglycosylation by glycosidase (galactosidase)

would be an alternative to this chemical treatment, but the removal of galactose

residues was incomplete in a preliminary experiment carried out by lab1 (data not

shown).

The major advantages of glycopeptide analysis are clearly illustrated in this

pilot study as follows. First, the average or maximum/minimum number of attached

sites can be counted. Second, relative abundances of different glycoform

compositions can be readily established at high sensitivity. Third, MS of

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glycopeptides does not miss the GalNAc-α-O-Ser/Thr (Tn epitope), which is

characterized by components having a higher number of HexNAc than Hex such as

5N4H or 5N3H.

Chromatographic Analysis of Tagged O-glycans

Chromatography of reductively aminated oligosaccharides carrying fluorescent

“tags” at their reducing ends is a well established procedure for quantitating mixtures

of N-glycans. It remains a popular method despite issues of variance between

laboratories (1) because it does not require highly sophisticated and expensive

instrumentation. Exploitation of this type of methodology in the O-glycan field is far

less common because free reducing sugars are required for the tagging reaction and

no tools are available that are capable of liberating O-glycans efficiently and cleanly

from glycopeptides or glycoproteins whilst retaining their reducing sugars.

Unfortunately no broad spectrum O-glycanase has yet been discovered. Therefore the

release of O-glycans requires base catalyzed chemical elimination and it has been

known for over forty years that the core 1 arm of O-glycans is readily “peeled” from

the 3-position of the reducing GalNAc residue under basic conditions unless the latter

is reduced. Hence reductive elimination is the only way of obtaining an artifact-free

O-glycan population. Nevertheless chromatographic/tagging methodologies can play

a useful role in O-glycan analysis, for example in the analysis of complex mucins (17),

provided peeling products and other chemical artifacts are taken into account in data

analysis. In the present study three laboratories (2,7,8) analyzed base-eliminated

fluorescently tagged glycans and their data nicely encapsulate the issues that need to

be considered when such methods are employed. O-Glycans were released manually

with anhydrous hydrazine (labs 2 and 7) or automatically with lithium hydroxide (lab

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8) or were tagged with phenylhydrazine, 2-(2-aminobenzoylamino)-2-

hydrazinocarbonyl-ethanethiol (ABHE) or 2-aminobenzoic acid (AA), respectively.

The ABHE tagging procedure involved a nanoparticle immobilization/derivatization

step and subsequent release of the tagged O-glycans (see Supplementary Methods).

Workflows and protocols are given in Scheme 2 and Supplementary Methods and

chromatographic profiles are given in Supplementary Figures S8-S10. Comparative

quantitative data are shown in Table II. The stark discrepancies between the data sets

highlight the artifact problems mentioned earlier. Nevertheless where replicate

experiments were carried out reasonable consistency was observed, so it can be

concluded that comparative chromatographic profiling is a valid technique under

carefully controlled conditions.

Mass Spectrometric Analysis of Reduced O-glycans

Nine labs analyzed reductively eliminated O-glycans using three general MS

strategies: (i) positive ion mode MALDI-MS fingerprinting supplemented by MALDI

and/or ESI-MS/MS sequencing of mixtures of permethylated glycans (labs 1, 9-12);

(ii) negative ion mode ESI-MS fingerprinting and ESI-MS/MS sequencing of native

glycans which were purified and separated by graphitized carbon on-line LC (labs

5,13,14); (iii) negative and positive ion mode MALDI-FTICR-MS of mixtures of

native glycans without on-line LC purification (lab 15).

Workflows and protocols are given in Scheme II and Supplementary Methods,

respectively, and typical data are shown in Supplementary Figures S11 and S12.

Quantitative information was extracted from MALDI fingerprints and LC-MS profiles

by measuring peak heights and peak areas, respectively. With one exception (lab 13,

see Supplementary Methods) no corrections were made for differences in the response

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factors of the various glycans. Exemplar quantitative data are shown in Table III for

permethylated and native samples, respectively, and data from all eight labs are

collated in Supplementary Table I. Collectively, these data are broadly in line with

the conclusions of the glycopeptide profiling experiments (see earlier), namely that

most of the glycans are core 1 type with monosialylated core 1 dominating in Sap-II

and non-sialylated core 1 being the major glycan in VDS and NUD. For technical

reasons the neutral glycans were missed in some of the on-line experiments (see

Supplementary Table 1). On-line graphitized carbon LC will not detect the Tn

antigen as single monosaccharides are not retained. Minor core 2 glycans were most

reliably observed in the analyses of permethylated samples (Supplementary Figs. S11

and S13 for confirmation via tandem MS). A weakness of both methodologies is that

monosaccharides such as Tn are difficult to analyze reliably because of masking by

matrix peaks in MALDI data and early elution together with impurities in on-line LC-

MS.

In summary, the study has shown that both (i) and (ii) are reliable strategies for

semi-quantitative O-glycan analysis except for the analysis of Tn. On the other hand,

strategy (iii), in which native samples were analyzed as mixtures by MS without

permethylation or on-line LC purification, gave equivocal data. Notably, spectra

were characterized by dominant matrix and artifact peaks and only one molecular ion

species was assignable to an authentic IgA O-glycan (see Supplementary Fig. S14). It

was also observed in some experiments that small quantities of glycopeptides (with

two or more amino acids) are recovered from reductive elimination reactions and their

presence may need to be taken into account when data are analyzed.

CONCLUSIONS

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In this study the O-glycan content of three IgA1 samples were analyzed by

fifteen participating laboratories. Six of these laboratories used either MALDI or ESI

to obtain mass fingerprints of the hinge glycopeptide (which has up to five occupied

O-glycosylation sites), three laboratories eliminated the O-glycans without reduction

and then tagged the free reducing ends prior to chromatographic analysis, nine

laboratories used reductive elimination to release the glycans which were then

analyzed by MS after permethylation (five laboratories), or as native glycans by LC-

MS (three laboratories) or MS alone (one laboratory). The main conclusions that can

be drawn from the study are summarized below.

Tryptic glycopeptides that were purified using comparable chromatographic

procedures gave broadly similar mass fingerprints irrespective of whether MALDI or

ESI was used. It was noted, however, that the more highly sialylated glycoforms

were somewhat less abundant in the MALDI fingerprints suggesting some loss of

sialic acid during the analysis. Nevertheless the fact that most of the sialic acid was

preserved demonstrates that MALDI is a suitable tool for profiling sialylated

glycoforms provided the linear ion mode is employed in the TOF analysis. Semi-

quantitative assignment of total glycan compositions deduced from ion abundances in

the mass fingerprints enabled inter-sample comparisons with respect to heterogeneity

and overall levels of sialylation. The likely presence of the Tn epitope in some of the

glycoforms was revealed by compositions rich in HexNAc. The compositional data

gives clues to site occupancy: for example the number of sites occupied cannot be

greater than the number of HexNAc residues in the glycoform composition, while

detailed profiling by MS of glycopeptides requires prior knowledge of the glycan

structures obtained by other methods such as chromatography of tagged glycans,

supplementary experiments employing, for example. Electron capture dissociation

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(ECD) or electron transfer dissociation MS/MS instrumentation are capable of

defining precise glycosylation sites as well as the size or suggested structures of

glycans at specific sites (6, 7, 18). Indeed, FTMS equipped with ECD has also been

used for structure determination of O-glycopeptides and shows potential for site

analysis (18, 19).

Chromatographic/tagging methodologies employed by three laboratories in this

study were found to give a diverse body of data that could not be readily reconciled

with the outputs of the more rigorous glycopeptide analyses described above. It was

clear from these experiments that peeling during non-reductive alkaline release of O-

glycans from glycopeptides, exacerbated by the frequent occurrence of artifactual

chromatographic peaks, severely impacts on the reliability of these methodologies for

quantitative O-glycomics. It was also apparent that neither automation of glycan

release nor the exploitation of nanoparticle technology for capturing the released

glycans has improved the efficacy of these methodologies.

On the other hand, there was good consistency amongst the data derived from

samples that were prepared by reductive elimination. Two general strategies were

shown to be highly effective for the reproducible analysis of reduced O-glycans: (i)

MALDI mass fingerprinting complemented by MALDI or ESI MS/MS sequencing of

mixtures of permethylated glycans, (ii) on-line LC-ESI-MS in the negative ion mode

of native samples. The first afforded the highest sensitivity detection of minor

components. Both methodologies are routinely semi-quantitative making them ideal

for comparative glycomics. Absolute quantification is also achievable with each

technology but requires well characterized standards for calibration of mass

spectrometry response factors. The lack of readily available quantified glycan

standards remains a logjam for quantitative glycomics.

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O-glycans of IgA are relatively simple, lack isomers and are not densely

clustered as normally observed in the mucins. This makes analysis of the

glycopeptides after proteolytic degradation ideal. However, in the mucins with more

complex and longer oligosaccharides this approach will be less useful. The O-glycans

have then to be alkaline released. Permethylation followed by direct MALDI-MS and

separation by liquid chromatography coupled to ESI-MS analysis are the two methods

of choice for the structural characterization of O-glycans in complex mixtures. The

permethylation methods employed in this study are suitable for neutral and sialic

acid-containing compounds, but need to be modified for sulfated oligosaccharides

(20).

The IgA from patients with multiple myeloma is derived from a B-cell clonal

population that tends to be almost monoclonal with the progression of the disease.

Although the glycosylation of myeloma proteins will thus be more homogenous than

is observed in polyclonal IgA from healthy individuals, the O-glycans exhibit a

degree of heterogeneity specific for each myeloma protein (7, 10). The differences

observed here probably reflect varying regulation of O-glycosylation in the different

B-cells clones. The distribution of O-glycan heterogeneity in NUD, VDS and SAP-II

is comparable to published analysis of myeloma IgA1 from other sources (7).

Acknowledgments -- The work was supported by the Swedish Research Council

(#7461 and equipment), the Swedish Cancer Foundation, MIVAC, IngaBritt and Arne

Lundberg Foundation, and Knut and Alice Wallenberg Foundation (GCH). N.K.

would like to thank Dr. Satsuki Itoh and Dr. Noritaka Hashii (National Institute of

Health Sciences, Tokyo, Japan). K-H.K. is supported by NSC 96-3112-B-001-018

17

(for the NRPGM Core Facilities). M.B.R. and J.N. would like to thank Stacy Hall

and Stephanie B. Wall for technical support. J.N. and M.B.R. are supported in part by

grants from National Institutes of Health DK077279, DK078244, DK80301,

DK71802, DK082753, and DK064400. Y.W. is supported in part by a grant from

Takeda Science Foundation. A.D., S.M.H., B.T. and K.C. acknowledge the support

of a Biotechnology and Biological Sciences Research Council (BBSRC) core grant

and a BBSRC Professorial Fellowship (to A.D.). A part of this work was supported

by the Global COE (Centers of Excellence) Program of Osaka University from the

Japan Society for the Promotion of Science (JSPS) and by the JSPS Core-to-Core

program.

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Tomana, M., Tomino, Y., Mestecky, J., and Novak, J. (2009) Aberrantly

glycosylated IgA1 in IgA nephropathy patients is recognized by IgG

antibodies with restricted heterogeneity. J Clin Invest 119, 1668-1677

14. Kobayashi, K., Vaerman, J. P., and Heremans, J. F. (1973) J-chain

determinants in polymeric immunoglobulins. Eur J Immunol 3, 185-191

15. Mestecky, J., Hamilton, R. G., Magnusson, C. G., Jefferis, R., Vaerman, J. P.,

Goodall, M., de Lange, G. G., Moro, I., Aucouturier, P., Radl, J.,

20

Cambiaso, C., Silvain, C., Preud'homme, J. L., Kusama, K., Carlone, G. M.,

Biewenga, J., Kobayashi, K., Skvaril, F., and Reimer, C. B. (1996) Evaluation

of monoclonal antibodies with specificity for human IgA, IgA subclasses and

allotypes and secretory component. Results of an IUIS/WHO collaborative

study. J Immunol Methods 193, 103-148

16. Wada, Y., Tajiri, M., and Yoshida, S. (2004) Hydrophilic affinity isolation and

MALDI multiple-stage tandem mass spectrometry of glycopeptides for

glycoproteomics. Anal Chem 76, 6560-6565

17. Xia, B., Royall, J. A., Damera, G., Sachdev, G. P., and Cummings, R. D.

(2005) Altered O-glycosylation and sulfation of airway mucins associated

with cystic fibrosis. Glycobiology 15, 747-775

18. Mirgorodskaya, E., Roepstorff, P., and Zubarev, R. A. (1999) Localization of

O-glycosylation sites in peptides by electron capture dissociation in a Fourier

transform mass spectrometer. Anal Chem 71, 4431-4436

19. Mormann, M., Paulsen, H., and Peter-Katalinic, J. (2005) Electron capture

dissociation of O-glycosylated peptides: radical site-induced fragmentation of

glycosidic bonds. Eur J Mass Spectrom (Chichester, Eng) 11, 497-511

20. Mitoma, J., Bao, X., Petryanik, B., Schaerli, P., Gauguet, J. M., Yu, S. Y.,

Kawashima, H., Saito, H., Ohtsubo, K., Marth, J. D., Khoo, K. H., von

Andrian, U. H., Lowe, J. B., and Fukuda, M. (2007) Critical functions of N-

glycans in L-selectin-mediated lymphocyte homing and recruitment. Nat

Immunol 8, 409-418

21

Table, Scheme and Figure Legends

Table I. Summary of the methodology/instrumentation employed in this study.

Notes: AE: anion exchange; AGC: AutoGlycoCutter; β-elim: beta-elimination; Ca:

graphitized carbon column, Gp: glycopeptide; HPLC: high performance liquid

chromatography; Hy: hydrazinolysis; Jac: affinity chromatography on Jacalin; OS:

oligosaccharides; Pe: permethylation; Pn; PNGase F treatment, N-glycan removal;

Pr: proteolytic digestion; RA: reduction and cysteine derivatization; RP: reverse

phase; Sep: chromatography Sepharose CL4B

Table II. Comparative data obtained by labs 2, 7 and 8 using chromatography

analyses.

Keys to symbols - yellow square: N-acetylgalactosamine, blue square: N-

acetylglucosamine, yellow circle: galactose, pink diamond: N-acetylneuraminic acid.

Table III. O-glycans comparative results obtained by lab 9 (MALDI-TOF MS) and

13 (LC-ESI MS).

Notes: *Combination of the two isomers; [Md+Na]+ corresponds to the mass of a

deutero-reduced permethylated glycan analyzed in positive mode (see Supplementary

Methods for lab 9), [M-H]- corresponds to the mass of a native glycan analyzed in

negative mode. For keys see Table II legend.

Scheme 1. Workflow of the various methodologies used for the O-glycopeptide

structural analysis.

Scheme 2. Workflow of the various methodologies used for the O-glycan structural

22

analysis.

Figure 1. MALDI profiles of IgA O-glycopeptides of Sap-II, VDS and NUD samples

(lab 1).

Spectra were acquired using the linear positive mode. Putative structures were deduced

from the molecular mass of glycopeptides and known structures of core 1 O-glycans for the

IgA1 hinge region. Average mass was used for these measurements, and mass accuracy was

± 1 Da. N, H and NA represent HexNAc (GalNAc), Hex (Gal) and Neu5Ac (N-

acetylneuraminic acid), respectively.

Figure 2. ESI mass spectra of IgA O-glycopeptides of Sap-II, VDS and NUD samples

(lab 2).

(a) Deconvoluted spectra of three samples. Monoisotopic masses of [M+H]+ ions are

indicated in the mass spectrum of Sap-II. (b) Real ESI mass spectrum of Sap-II

before deconvolution. Charge state of each peptide ion signal is given in parentheses.

Samples were analyzed by LC-MS in positive ion mode using a Paradigm MS4 HPLC

system coupled to a Thermo FT-ICR mass spectrometer. Mass accuracy was ± 0.1 Da.

N, H and NA represent HexNAc (GalNAc), Hex (Gal) and NeuAc (N-

acetylneuraminic acid), respectively.

Figure 3. Comparison of the glycan profiles obtained by laboratories having used MS

of glycopeptides as analytical tool.

Relative abundance of glycan structures were calculated from the signal intensities in

the mass spectra of Sap-II (Figs 1 and 2 and supplementary Figs. 1 and 2). Error bars,

2 SD. Protonated ions were analysed. MS mode was MALDI linear-TOF in lab1,

23

and ESI LTQ-FT-ICR, ESI Orbitrap and ESI Q-TOF in labs 2, 4, and 5, respectively.

IonSpec FT-ICRMALDI-MS, ESI-MSOS / Pn, β-elimLab 15

Agilent 3D Ion TrapOn-line LC ESI-MSOS / RA, SDS-PAGE, β-elimLab 14

Thermo LTQOn-line Ca-LC ESI-MSOS / SDS-PAGE, β-elimLab 13

MALDIPe, MALDI-MSOS / β-elimLab 12

ABI 4700 Proteomics analyzerThermo LCQ-MS

Pe, MALDI-MS, ESI-MSOS / β-elimLab 11

ABI 4700 Proteomics analyzerWaters MALDI Q-TOF Ultima

Pe, MALDI-MSOS / β-elimLab 10

Bruker-Daltonik GmbH Reflex IV MALDI-TOFShimadzu AXIMA-QIT MALDI Quadrupole Ion Trap TOF

Pe, MALDI-MSOS / Pn, β-elimLab 9

ABI Voyager DE Pro MALDI-TOF Shimadzu LC MS-IT TOF

2-AA, HPLC, MALDI-MS, On-line LC ESI-MSOS / AGCLab 8

-ABHE, HPLCOS / Hy, BlotglycoLab 7

Thermo LTQ-FT-ICR On-line RP-LC ESI-MSGp / RA, SDS-PAGE, PrLab 6

Waters Ultima Q-TOF Bruker HCT Ultra PTM Discovery System

On-line AE-LC ESI-MSOn-line Ca-LC ESI-MS

Gp / RA, SDS-PAGE, PrOS / RA, SDS-PAGE, β-elim

Lab 5

Thermo OrbitrapSep, On-line RP-LC ESI-MSGp / RA, Pr, PnLab 4

Thermo LCQ Deca XPJac, HPLC, On-line RP-LC ESI-MSGp / RA, PrLab 3

Thermo LTQ-FT-ICR On-line RP-LC ESI-MSOn-line Ca-LC ESI-MS

Gp / RA, Pn, PrOS / Hy

Lab 2

ABI Voyager MALDI-TOF DE ProThermo LTQ with ETD

Sep, MALDI-MS, ESI-MSPe, MALDI-MS

Gp / RA, PrOS / β-elim

Lab 1

MS instrumentAnalysis strategyAnalyte / preparationLab

AE: anion exchange; AGC: AutoGlycoCutter; β-elim: beta-elimination; Ca: graphitized carbon column, Gp: glycopeptide; HPLC: high performance liquid chromatography; Hy: hydrazinolysis; Jac: affinity chromatography on Jacalin; OS: oligosaccharides; Pe: permethylation; Pn; PNGase F treatment, N-glycan removal; Pr: proteolytic digestion; RA: reduction and cysteine derivatisation; RP: reverse phase; Sep: chromatography sepharose CLB4

Table I. Summary of the methodologies/instrumentation employed

6.82.821.28.113.3

19.57.069.265.471.058.437.136.5

24.443.461.128.231.926.7

3.5

7.712.212.414.411.2

100 100 100 100 100 100 100 100 100

7.64.5

2.6

Sap II

2.7

VDS

2.3

NUD

Lab 7 Lab 8Lab 2O-linked glycan

9.526.7Unidentified component

total

32.26.4

24.78.040.439.0

Sap IIVDSNUDSap IIVDSNUDStructure

Table II. Comparative data obtained by labs 2, 7 and 8 using chromatography analyses

(%)

LC-ESI MSMALDI-TOF MSO-linked glycan

1.0--0.4-0.7

100total

(%)

100 100 100 100 100

[Md+Na]+ 1346 [M-H]- 1040

7.08.06.01.3-0.7[Md+Na]+ 1257 [M-H]- 966

---0.3-2.3[Md+Na]+ 984 [M-H]- 749

-1.01.0--[Md+Na]+ 896 [M-H]- 675

60.024.023.077.6

23.0*

26.3[Md+Na]+ 896[M-H]- 675

32.066.070.020.477.070.0[Md+Na]+ 535 [M-H]- 384

Sap IIVDSNUDSap IIVDSNUD(m/z)Structure

[Md+Na]+ corresponds to the mass of a deutero-reduced permethylated glycan analyzedin positive mode (see Supplementary Methods for lab 9). [M-H]- corresponds to the massof a reduced native glycan analyzed in negative mode (see Supplementary Methods for lab 13).*Combination of the two isomers.

Table III. O-glycans comparative results obtained by lab 9 (MALDI-TOF MS) and 13 (LC-ESI MS)

Scheme 1 Workflow of glycopeptide analysisLaboratory number is indicated in parentheses (see also Table I).

Glycopeptides

Enrichment / Chromatography

(1,3,4)

Sepharose CL4B Jacalin, HPLC

MALDI-MS On-line LC ESI-MS

(1,4) (3)

(3,4)

(1)

(2,5,6)

ESI-MS

Reducing treatment Cysteine derivatization

N-glycan removal

(1,3,4)(2)

Proteolytic digestion

NUD / VDS / Sap-II

)6,5()4,3,2,1(

SDS-PAGE

Neuraminidase

(6) (5,6)

Samplehandling

Enrichment

Analysis

N-glycan removal(4) (1,2,3,5,6)

4000 700060005000 8000

Sap-II

4000 700060005000 8000

4000 700060005000 8000

NUD

VDS

m/z

3H3N

1NA

1 H1N

4H4N

1NA

4 H5N

2NA

4 H4N

2NA

4H5N

1NA

4H4N

3NA

4H5 N

3NA

4H5 N

4NA

2H2N

2H3N

4 H4N

4 NA

3H4N

4H4N

4H5N

3H3N

3H4N

2H2N

4 H4N

4H4 N

1NA4H

5N

5H5N

4H5N

1NA

4H5N

1NA

3H3N

3H4N

4 H4N

4H4N

1NA

4H5N

5H5N

4 H5 N

1NA

4 H4 N

2NA

Rel

ativ

e In

tens

ity

100

0

100

0

100

0

Figure 1

(543

8.8)

(560

0.9)

(580

4.1) (5

892.

2)

(609

5.4)

(618

3.4)

(638

6.6)

(647

4.7)

(667

7.9)

(676

5.9)

(696

9.1)

Figure 2

(a)

(b)

NUD

5000 6000 7000

Rel

ativ

e ab

unda

nce

100

0

VDS

Rel

ativ

e ab

unda

nce

100

05000 6000 7000

Rel

ativ

e ab

unda

nce

100

05000 6000 7000

Sap- II

m/z

4N3H

4N3H

4N3H

3N3H

3N3H

4N2H

3N3H

1NA

4N4H

4N4H

4N4H

5N3H

5N3H

4N3H

1NA

4N3H

1NA

4N3H

1NA

5N4H

5N4H

5N4H

3N3H

2NA

3N3H

2NA

6N3H

6N3H

4N4H

1NA

4N4H

1NA

4N4H

1NA

5N3H

1NA

5N3H

1NA

5N5H

5N5H

5N5H

6N4H

6N4H

6N4H

4N3H

2NA

4N3H

2NA

5N4H

1NA

5N4H

1NA

5N4H

1NA

6N3H

1NA

4N4H

2NA

4N4H

2NA

4N4H

2NA

6N3H

1NA

5N3H

2NA

5N5H

1NA

5N5H

1NA

5N3H

2NA

6N4H

1NA

4N3H

3NA

5N4H

2NA

5N4H

2NA

5N4H

2NA

6N4H

1NA

6N3H

2NA

6N5H

1NA

6N5H

1NA

4N4H

3NA

4N4H

3NA

5N3H

3NA

5N5H

2NA

6N4H

2NA

5N4H

3NA

5N4H

3NA

5N5H

2NA

4N4H

4NA

5N5H

3NA

5N4H

4NA

5N4H

4NA

5N5H

4NA

Hex 5

HexN

Ac5N

euAc

4(4)

Hex 4

HexN

Ac6N

euAc

2(4)

Hex 3

HexN

Ac5N

euAc

3(4)

Hex 3

HexN

Ac6N

euAc

2(4)

Hex 3

HexN

Ac4N

euAc

3(4)

Hex 4

HexN

Ac5N

euAc

4(5)

Hex 4

HexN

Ac5N

euAc

3(5)

Hex 3

HexN

Ac4(4

) Hex 4

HexN

Ac4N

euAc

1(4)

Hex 4

HexN

Ac4(4

)

Hex 4

HexN

Ac5(4

) Hex 5

HexN

Ac5(4

)

Hex 4

HexN

Ac5N

euAc

1(4)

Hex 4

HexN

Ac4N

euAc

2(4)

Hex 5

HexN

Ac5N

euAc

1(4)

Hex 4

HexN

Ac4(3

)

Hex 3

HexN

Ac4(3

)

Hex 4

HexN

Ac5N

euAc

4(4)

Hex 4

HexN

Ac4N

euAc

4(4)He

x 4He

xNAc

4Neu

Ac3(4

)

Hex 3

HexN

Ac4N

euAc

1(4)

Hex 3

HexN

Ac5(4

)

Hex 2

HexN

Ac4(4

)He

x 3He

xNAc

3(4)

Hex 4

HexN

Ac4N

euAc

2(5)

Hex 4

HexN

Ac5N

euAc

1(5)

Hex 4

HexN

Ac4N

euAc

1(5)

Hex 4

HexN

Ac4(5

)

Hex 3

HexN

Ac4(5

)

Hex 4

HexN

Ac5N

euAc

2(4)

Hex 4

HexN

Ac5N

euAc

3(4)

Hex 5

HexN

Ac5N

euAc

2(4)

Hex 4

HexN

Ac6N

euAc

1(4)

Hex 3

HexN

Ac5N

euAc

2(4)

Hex 3

HexN

Ac6N

euAc

1(4)

Hex 3

HexN

Ac4N

euAc

2(4)

Hex 3

HexN

Ac5N

euAc

1(4)

Hex 3

HexN

Ac3N

euAc

2(4)

Hex 4

HexN

Ac4N

euAc

3(5)

Hex 4

HexN

Ac5N

euAc

2(5)

Hex 3

HexN

Ac4N

euAc

1(5)

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

Relat

ive a

bund

ance

0

50

Hex 4

HexN

Ac4N

euAc

4(5)

m/z

Hex 5

HexN

Ac5N

euAc

3(4)

Hex 4H

exNA

c 6(4)

Hex 5

HexN

Ac6N

euAc

1(4)

Hex 3

HexN

Ac6(4

)

Hex 4

HexN

Ac5(5

)

Hex 5

HexN

Ac5N

euAc

4(4)

Hex 4

HexN

Ac6N

euAc

2(4)

Hex 3

HexN

Ac5N

euAc

3(4)

Hex 3

HexN

Ac6N

euAc

2(4)

Hex 3

HexN

Ac4N

euAc

3(4)

Hex 4

HexN

Ac5N

euAc

4(5)

Hex 4

HexN

Ac5N

euAc

3(5)

Hex 3

HexN

Ac4(4

) Hex 4

HexN

Ac4N

euAc

1(4)

Hex 4

HexN

Ac4(4

)

Hex 4

HexN

Ac5(4

) Hex 5

HexN

Ac5(4

)

Hex 4

HexN

Ac5N

euAc

1(4)

Hex 4

HexN

Ac4N

euAc

2(4)

Hex 5

HexN

Ac5N

euAc

1(4)

Hex 4

HexN

Ac4(3

)

Hex 3

HexN

Ac4(3

)

Hex 4

HexN

Ac5N

euAc

4(4)

Hex 4

HexN

Ac4N

euAc

4(4)He

x 4He

xNAc

4Neu

Ac3(4

)

Hex 3

HexN

Ac4N

euAc

1(4)

Hex 3

HexN

Ac5(4

)

Hex 2

HexN

Ac4(4

)He

x 3He

xNAc

3(4)

Hex 4

HexN

Ac4N

euAc

2(5)

Hex 4

HexN

Ac5N

euAc

1(5)

Hex 4

HexN

Ac4N

euAc

1(5)

Hex 4

HexN

Ac4(5

)

Hex 3

HexN

Ac4(5

)

Hex 4

HexN

Ac5N

euAc

2(4)

Hex 4

HexN

Ac5N

euAc

3(4)

Hex 5

HexN

Ac5N

euAc

2(4)

Hex 4

HexN

Ac6N

euAc

1(4)

Hex 3

HexN

Ac5N

euAc

2(4)

Hex 3

HexN

Ac6N

euAc

1(4)

Hex 3

HexN

Ac4N

euAc

2(4)

Hex 3

HexN

Ac5N

euAc

1(4)

Hex 3

HexN

Ac3N

euAc

2(4)

Hex 4

HexN

Ac4N

euAc

3(5)

Hex 4

HexN

Ac5N

euAc

2(5)

Hex 3

HexN

Ac4N

euAc

1(5)

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 20001000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

Relat

ive a

bund

ance

0

50

Hex 4

HexN

Ac4N

euAc

4(5)

m/z

Hex 5

HexN

Ac5N

euAc

3(4)

Hex 4H

exNA

c 6(4)

Hex 5

HexN

Ac6N

euAc

1(4)

Hex 3

HexN

Ac6(4

)

Hex 4

HexN

Ac5(5

)

(543

5.37

)

(559

7.42

)(5

638.

45)

(572

6.46

)

(580

0.50

)(5

814.

48)

(584

1.52

)(5

888.

51)

(592

4.54

)(5

962.

55)

(600

3.58

)(6

017.

56)

(609

1.59

)(6

132.

62)

(617

9.61

)(6

220.

64)

(625

3.65

)(6

294.

67)

(630

8.65

)

(638

2.69

)(6

423.

72)

(645

6.73

)(6

470.

71)

(651

1.73

)(6

544.

74)

(658

5.77

)

(667

3.78

) (676

1.80

)

(696

4.88

)

(712

6.93

)

Scheme 2 Workflow of O-glycan analysisLaboratory number is indicated in parentheses (see also Table I).

(5,13,14)BlotglycoABHE

HPLC

(7,8)

Derivatization(7) (8)

2-AA tagging

MALDI-MS and/or MS/MS

On line ESI-MS and/or MS/MS

(8)

(8)

Permethylation

(1,2,7,8,9,10,11,12) (5,13,14,15)

Hydrazinolysis

(15)

Native analysis

(11)

Samplehandling

Derivatization

Analysis

O-glycans

(2,7) (1,8)

Automated releaseBeta-elimination

(10,11,12)

Reduction-alkylation Trypsin

(9,15)

N-glycans removal

(1)

(8)

(5,14)

SDS-PAGE

(13)

Reduction Alkylation

(1,9,10,11,12)

(1,9,10,11,12)

MALDI-MS ESI-MS

(5,11,13,14)

(15)

NUD / VDS / Sap-II

(2)Phenylhydrazine

(2)

Reduction

1.2

0.2

0.4

0.6

0.8

1.0

rela

tive

abun

danc

e

lab 1lab 2lab 4lab 5

4N3H

4N4H

5N4H

4N4H

1NA

5N4H

1NA

4N4H

2NA

5N4H

2NA

4N4H

3NA

5N4H

3NA

4N4H

4NA

5N4H

4NA

structures

0.0

Figure 3