How to Bring the “Unseen” Proteome to the Limelight via Electrophoretic PreFractionation...

How to Bring the ‘‘Unseen’’ Proteome to the Limelight viaElectrophoretic Pre-Fractionation Techniques

Pier Giorgio Righetti,1,4 Annalisa Castagna,1 Ben Herbert,2 and Giovanni Candiano3

The present review reports a panoply of electrophoretic methods as pre-fractionation toolsin proteomic investigations in preparation for mass spectrometry or two-dimensional elec-trophoresis map analysis. Such electrophoretic pre-fractionation protocols include all thoseelectrokinetic methodologies which are performed in free solution, most of them relying onisoelectric focusing steps (although some approaches based on gels and granulated mediaare also discussed). Devices associated with electrophoretic separations are multi-chamberapparatuses, such as the multi-compartment electrolyzers equipped with either isoelectricmembranes or with isoelectric beads, Off-Gel electrophoresis in a multi-cup device and theRotofor, an instrument also based on a multi-chamber system but exploiting theconventional technique of carrier-ampholyte-focusing. Other free-flow systems, as well asminiaturized chambers, are also described.

KEY WORDS: Proteomics; pre-fractionation; isoelectric focusing; two-dimensional maps.

ABBREVIATIONS: FFE: free-flow electrophoresis; FF-IEF: free-flow isoelectric focusing;IPG: immobilized pH gradients; CBI: codon bias index; EOF: electroendoosmotic flow;2-DE: two-dimensional electrophoresis.

INTRODUCTION

Although, at the latest count, the total number of coding genes in humans wouldappear to oscillate between only 25,000 and 30,000 (Southan, 2004), the complexityof the human proteome could, nevertheless, be overwhelming. An example on such avast complexity and thus on the stringent need for pre-fractionation in proteomeanalysis comes from some recent articles on the plasma proteome (Anderson andAnderson, 2002; Pieper et al., 2003). If one assumes that there are just 500 true‘‘plasma proteins’’, each present in 20 variously glycosylated forms and in five dif-ferent sizes, one would end up with 50,000 molecular forms. If one furtherhypothesizes that the ca. 30,000 gene products in the human proteome exist onaverage as 10 splice variants, cleavage products and post-translational modifications,

1Department of Industrial and Agricultural Biotechnologies, University of Verona, Strada Le Grazie 15,

37134, Verona, Italy.2Proteome Systems, 35 Waterloo Rd, North Ryde, 2113, Sydney, NSW, Australia.3Laboratory of Physiopathology of Uremia, G. Gaslini Children’s Hospital, 16148, Genova, Italy.4To whom should be addressed. E-mail: [email protected]

Bioscience Reports, Vol. 25, Nos. 1/2, February/April 2005 (� 2005)

DOI: 10.1007/s10540-005-2844-2

3

0144-8463/05/0400-0003/0 � 2005 Springer Science+Business Media, Inc.

this would yield some additional 300,000 protein forms. Rammensee (2004) reportedthat, by splicing proteins into small pieces, stitching different portions together, andthen cutting out amino acids sequences from the melded pieces, cells can manufac-ture a very large variety of new forms from the original gene coded proteins.Moreover single proteins such as antibodies might contain more than 1,000,000different epitopes sequences which add to the complexity of the serum content. Tothis very intricate situation, additional difficulties come from the fact that thedynamic range, at least in serum, might be more than 10 orders of magnitude.

In essence it is clear that a natural way to analyze the content of a proteome oreven checking phenotyping differences, is to pre-fractionate the proteome into dis-crete groups and then analyze separately each group. Some recent papers suggestthat a problem of this vast complexity could be overcome not by pre-fractionationbut rather by running a series of narrow-range IPG strips (covering no more than 1pH unit). Hoving et al. (2000) and Westbrook et al. (2001) note that ‘‘zoom’’ and‘‘ultra-zoom’’ gels are quite important for avoiding or at least minimizing theproblem of spot overlapping in 2-DE, an ever present hazard in 2-DE maps (seePietrogrande et al., 2002, 2003; Campostrini et al., 2005). The use of large-size gelslabs (18-cm or longer in the first dimension, 18 · 20 cm, or larger, in the seconddimension), would even dramatically increase the resolution, as reported by Corthalset al. (2000) and Wildgruber et al. (2000). By that way it seems possible to makeportions of bi-dimensional mappings without the necessity of pre-fractionating thesample. The entire, wide-range map would then be electronically reconstructed bystitching together the narrow-range maps. In practice, however, it remains the factthat, even when using very narrow IPG strips, they have to be loaded with the entiretissue lysate with consequent massive precipitation, along with the additionaldrawback that the proteins which should focus in the chosen narrow-range IPGinterval will be strongly under-represented because they will be only a small fractionof the entire sample loaded (Herbert et al., 2004). These serious issues have beendebated in a recent work by Gygi et al. (2000), who argued that, in 2D gels, proteinsfrom genes with codon bias values of <0.1 (low-abundance species), large-sizeproteins (>100 kDa) and most membrane proteins could not be found.

Several published papers highlight major limitations of available technologiesfor proteome investigations. Current approaches are qualified as incapable of havinga whole vision of the proteome, even limited to structural aspects. For instancestrongly alkaline proteins are poorly represented when using classical two-dimen-sional electrophoresis, as underlined by Bae et al. (2003), and highly hydrophobicproteins cannot be properly solubilized and consequently not analyzed and/oridentified. Electrophoresis-based methods taken alone (still the most commonly usedto date) are neither appropriate for polypeptides of masses lower than 5000 Da, noreffective for very alkaline proteins. Only mass spectrometry contributes significantlyto the analysis of low sized polypeptides. To this panel it is to be added that post-translational modifications and especially glycosylations are still part of the non-resolved dilemmas. In this situation authors estimate that only about 20�30% ofexpressed proteins are detectable by standard methods to date.

Pre-fractionation, in all of its possible variants, as here reviewed, appears to bethe logical way to follow in the attempt to make a step in the right direction. Aselegantly stated by Pedersen et al. (2003), in fact, pre-fractionation could be a

4 Righetti, Castagna, Herbert, and Candiano

formidable tool for ‘‘mining below the tip of the iceberg to find low abundance andmembrane proteins’’. So, let us wear our proper mining tools, a pickaxe, acetylenelamp, helmet and goggles and descend deep inside the mine to start our digging.Although several types of pre-fractionation tools exist, we will limit this review onlyto electrophoretic techniques. Centrifugal pre-fractionation, for isolation of cellorganelles, has been surveyed in a number of reports (Cordwell et al., 2000; Corthalset al., 2000; Jung et al., 2000; Dreger, 2003; Huber et al., 2003; Stannard et al.,2004). Chromatographic techniques have also been covered by several authorsincluding us (Lopez, 2000; Isaaq et al., 2002; Righetti et al., 2003a, 2003b; Lescuyeret al., 2004).

PREPARATIVE ZONE ELECTROPHORESIS

Although plain zone electrophoresis in gel cylinders would appear to be justabout the last resort, it was recently applied by Fountoulakis and Juranville (2003) tothe enrichment of low-abundance brain proteins, by eluting some 80 fractions (eachof 10 ml) from a 11%T gel equilibrated with 0.1% lithium dodecyl sulphate (LDS).Interestingly, they could enrich relatively low molecular mass proteins, such ashippocalcin, visinin, 14-3-3 proteins. By the same token, one could perhaps exploitthe electrosmotic-pump-driven apparatus of Hayakawa et al. (2003), although gel-based systems do not appear to be popular in pre-fractionation protocols.

CONTINUOUS ELECTROPHORESIS IN FREE LIQUID FILMS

Over gel phases, this technique has the advantage that much higher sampleloads can be applied, coupled to the absence of possible protein modifications in-duced by free monomers always present in gel phases (Chiari et al., 1992; Bordiniet al., 2000). Present equipment derives from the concepts and instrumentation ofHannig (1967, 1982), by which the electrolyte solution flows in a direction normal tothe lines of forces of the electric field and the mixture to be separated is addedcontinuously at a small spot in the flowing medium. Components of the mixture aredeflected in diagonal trajectories according to their electrophoretic mobility and canbe collected at the bottom of the chamber into as many as 96 fractions. Free-flowelectrophoresis (FFE) was born as a technique for purifying cells and sub-cellularorganelles, which could be recovered highly purified as thin zones, due to their verylow diffusion coefficients. The Hannig apparatus went through successive designsand improvements, from an original liquid descending curtain to the present com-mercial version, dubbed Octopus, exploiting an upward liquid stream (Kuhn andWagner, 1989). Instrumental to the success of the method, especially when run forlong periods of times, is the constancy of the elution profile at the collection port, sothat the same protein species is always collected into the same test tube. Thus,electroendoosmotic flow should be suppressed via a number of ways, including glass-wall silanol deactivation and addition of polymers (e.g., 0.1% hydroxypropylmethylcellulose), providing dynamic wall coating and proper liquid viscosity. FFE in aProTeam apparatus was recently reported by Zischka et al. (2003) for purification ofS. cerevisiae mitochondria, previously purified by fractional centrifugation. Theseauthors claimed identification of many more proteins (n = 129) from FFE-purified

5How to Bring the ‘‘Unseen’’ Proteome to the Limelight

mitochondria as compared with mitochondrial protein extracts isolated by differ-ential centrifugation (n = 80). In addition, a marked decrease of degraded proteinswas found in the FFE-purified mitochondrial protein extracts, suggesting that theorganelles were contaminated by lysosomes.

FFE would not be ideal for protein pre-fractionation, though, due to theirhigher diffusion coefficients, as compared with cells and organelles. However,Kobayashi et al. (2003) reported a microfabricated FFE device useful for continuousseparation of proteins. Their separation chamber is barely 66 · 70 mm in size, with agap between the two Pyrex glass plates of only 30 lm (see Fig. 1a and b). The liquidcurtain and the sample are continuously injected from five and one holes, respec-tively, at the top. At the bottom of the chamber, a micromodule fraction collector,consisting of 19 stainless steel tubes, is connected perpendicular to the chamber andliquid stream. FFE, for protein separation, would work much better in the isoelectricfocusing mode (FF-IEF), due to built-in forces impeding entropic peak dissipation.The first report on the use of FF-IEF for pre-fractionation of total cell lysates fromHeLa and Ht1080 cell lines, in view of a subsequent 2-DE map, is perhaps the one ofBurggraf et al. (1995), who collected individual or pooled fractions for further 2-DEanalysis. Hoffman et al. (2001) proposed FF-IEF as the first dimension of a 2-DEmap, the eluted fractions being directly analyzed by orthogonal SDS-PAGE. In turn,individual bands in the second SDS-PAGE dimension were eluted and analyzed byelectrospray ionization, ion-trap MS. By this approach, they could identify a numberof cytosolic proteins of a human colon carcinoma cell line. One advantage of FF-IEF is immediately evident from their data: large proteins (e.g., vinculin, Mr116.6 kDa) could be well recovered and easily identified; on the contrary, recovery oflarge Mr species has always been problematic in IPG gels. Weber et al. (2004) havealso adopted FF-IEF for the efficient separation and analysis of peroxisomalmembrane proteins. Their success was documented by the detection of PMP22, themost hydrophobic and basic protein (pI>10) of peroxisomal membranes. Perhapspre-fractionation of proteins could also be attempted by IEF in the recently-revivedvortex-stabilized, free-flow electrophoretic device (Ivory, 2004; Tracy and Ivory,2004).

ROTATIONALLY STABILIZED FOCUSING APPARATUS: THE ROTOFOR

Behind this remarkable invention by M. Bier, there is a long history going backto the doctoral thesis of Hjerten (1967): as he was trained in astrophysics, hisapparatus was a ‘‘Copernican revolution’’ in electrokinetic methodologies. Hjertenwas the first one to propose electrophoretic separations in a free zone (i.e., in theabsence of anticonvective, capillary media, such as polyacrylamide and agarose gelnetworks) but he had to fight the noxious phenomenon of electrodecantation,induced by gravity. He thus devised rotation of the narrow-bore tubes used aselectrophoretic chambers around a horizontal axis, mimicking celestial planet mo-tions! This must have spurred the fantasy of Svensson-Rilbe, in those days a col-league at the Uppsala University, who finally described a large multi-compartmentelectrolyzer, capable of fractionating proteins in the gram range (Jonsson and Rilbe,1980). The cell was assembled from 46 compartments, accommodating a totalsample volume of 7.6 l, having a total length of 1 m, hardly user-friendly! Cooling


Fig. 1. Scheme of the miniaturized FFE apparatus. Reservoir R1 is used for the elec-

trophoresis buffer, whereas R2 (0.1 M aqueous NaOH�ethanol, 50:50, v/v), R3 (0.01 M

HCl) and R4 (80% aqueous ethanol) are used for washing cycles. S1 is the sample

reservoir, P1�P3 are peristaltic pumps for pouring the solutions into the separation

chamber, the electrode reservoirs and the sample port, respectively. D is a dumper, DRN

a drain duct, PF is a fuse unit for excessive pressure. B: Scheme of the micromodule

fraction separator (MFS). A cross-section of the separation chamber’s bottom with upper

and lower parts of Pyrex glass is shown (from Kobayashi et al., 2003, by permission).


and stirring were affected by slow rotation of the whole apparatus in a tank filledwith cold water. Bier’s 50-year-long love affair with preparative electrophoresis infree solution produced, as a last evolutionary step, a remarkable device, the Rotofor(Egen et al., 1988; Bier, 1998). A preparative-scale Rotofor is capable of beingloaded with up to 1 g of protein, in a total volume of up to 55 ml. A mini-Rotofor,with a reduced volume of about 18 ml, is also available. The device is assembledfrom 20 sample chambers, separated by liquid-permeable nylon screens, except at theextremities, where cation- and anion-exchange membranes are placed against theanodic and cathodic compartments, respectively, so as to prevent diffusion withinthe sample chambers of noxious electrodic products. At the end of the preparativerun, the 20 focused fractions are collected simultaneously by piercing a septum at thechambers’ bottom via 20 needles connected to a vacuum source. The narrow-pIrange fractions can then be used to generate conventional 2-DE maps. This is theoriginal approach described by Hochstrasser et al. (1991). In recent times, thismethodology has taken another turn: the Rotofor is used directly as the firstdimension of a peculiar 2-DE methodology, in which each fraction is further ana-lyzed by hydrophobic interaction chromatography, using non-porous reversed-phaseHPLC (Zhu et al., 2003). Each peak collected from the HPLC column is then di-gested with trypsin, subjected to MALDI-TOF MS analysis and MSFit databasesearching. By this approach, Wall et al. (2000) have been able to resolve a total of ca.700 bands from a human erythroleukemia cell line. It should be stated, though, thatthe pI accuracy of this methodology which is still based on conventional carrierampholyte-isoelectric focusing, CA-IEF, is quite poor: it ranges from ±0.65 to±1.73 pI units, a large error, indeed. On a similar line of thinking, Davidsson et al.(2001) have sub-fractionated human cerebrospinal fluid and brain tissue, whereasWang et al. (2002) have mapped the proteome of ovarian carcinoma cells. Morerecently, Xiao et al. (2004) have reported a novel application of the Rotofor, not justfor fractionation of intact proteins in presence of carrier ampholytes, but for frac-tionation of peptide digests of an entire proteome (in this case, human serum) in anampholyte-free environment. The peptides themselves would act as carrier ampho-lyte-buffers and create a pH gradient via an ‘‘autofocusing’’ process (with a caveat,though: the pH gradient will be quite poor, since only a few peptides have goodbuffering power and conductivity in the pH 5�8 range).

THE GRADIFLOW

The Gradiflow is a multi-functional electrokinetic membrane apparatus that canprocess and purify protein solutions based on differences of mobility, pI and size(Margolis et al., 1995; Horvath et al., 1996). Its interfacing with 2-DE map analysiswas demonstrated by Corthals et al. (1997), who adapted this instrument for pre-fractionation of native human serum and enrichment of protein fractions. In a morerecent report (Locke et al., 2002), this device was also shown to be compatible, in thepre-fractionation of bakers yeast and Chinese snow pea seeds total cellular extracts,with the classical denaturing/solubilizing solutions of 2-DE maps, comprising urea/thiourea and surfactants. Whereas, in the case of size fractionation, this can beachieved with polyacrylamide coated membranes at different %T and %C forsieving of macromolecules in given Mr ranges, its use for separating approximate pI


fractions is more complex and has to adopt low-conductivity buffers, such as thosedevised by Bier et al. (1984), so as to allow reasonably high voltage gradients andrelatively short separation times. It should be remembered, though, that the Gra-diflow operates on the principle of binary fractionations, so that, in general, only twopopulations can be collected during each run, the so called ‘‘upstream’’ and‘‘downstream’’ fractions. More recently, Bae et al. (2003) adopted the Gradiflow forpre-fractionation of alkaline proteins from Helicobacter pylori, although the termi-nology ‘‘extremely basic fraction’’ seems an exaggeration, considering that all thespecies identified by MALDI-TOF MS hardly reached pI values as high as pH 10.

SAMPLE PRE-FRACTIONATION VIA MULTI-COMPARTMENT

ELECTROLYZERS WITH ISOELECTRIC MEMBRANES

We have already mentioned multi-compartment electrolyzers (MCE; Jonssonand Rilbe, 1980; Bier, 1998), as a class of instruments based on conventional IEF inpresence of soluble, amphoteric buffers (carrier ampholytes, CA). However, theMCEs based on Immobiline membranes represent a quantum jump over the previ-ous technique (Righetti et al., 1989, 1990, 1992). This method relies on isoelectricmembranes, fabricated with the same acrylic monomers adopted in IPG fractiona-tions (Wenger et al., 1987; Righetti, 1990). Advantages of such a procedure areimmediately apparent: (i) such a device offers a method that is fully compatible withthe subsequent first dimension separation in 2-DE maps, a focusing step based onImmobiline technology. Thus, protein mixtures harvested from the various chambersof this apparatus can be loaded onto IPG strips without any need for furthertreatment, in that they are isoelectric and isoionic; (ii) it permits harvesting a pop-ulation of proteins having pI values precisely matching the pH gradient of anynarrow (or wider) IPG strip; (iii) as a corollary of the above point, much reducedchances of protein precipitation will occur, as compared to loading onto a narrowIPG strip an unfractionated sample composed of a much wider pI spectrum (in thelatter case, proteins non-isoelectric in the given pH range will massively precipitatetowards the ends of the IPG strips, most often co-precipitating neighbouring spe-cies); (iv) due to the fact that only proteins co-focusing in the same IPG interval willbe present, much higher sample loads can be operative, permitting detection of low-abundance proteins.

The original apparatus, as miniaturized by Herbert and Righetti (2000),Righetti et al. (2001) and Herbert et al. (2004), is shown schematically in Fig. 2a. Inthis exploded view, two terminal electrodic chambers are used to block, in between,three sample chambers. Fig. 2b is a schematic diagram of the MCE for initial plasmafractionation. The four disks in the upper part are the isoelectric membranes insertedin between the various chambers. In this particular set-up, the couple pI 5.0 and pI6.0 is used as a trap for capturing albumin. By properly exploiting this pre-frac-tionation device, Pedersen et al. (2003) have been able to capture and detect a largenumber of the ‘‘unseen’’ yeast membrane proteome.

Figure 3 gives an example of the large number of membrane proteins detected,via this pre-fractionation protocol, in the pH 7�10.5 range, an interval thatcartographers of the 16 century would have described as ‘‘terra incognita’’, devoid ofany landmarks, and stamped inside the contour of the map the inscription:


‘‘hic sunt leones’’, just as they did with the African continent. These data fullymisspell the notion expounded by Gygi et al. (2000) (see Introduction) that 2-DEmaps cannot detect membrane and low abundance proteins (see also Herbert et al.,2003); the key for ferreting them out is to use appropriate pre-fractionation methodsassociated with concentration.

A number of additional approaches have been described such as miniaturizeddevices (Zuo and Speicher, 2002) and the Rotofor accommodating isoelectricmembranes (Shang et al., 2003). Zhu and Lubman (2004) have modified the Iso-Prime device from Hoefer so as to lessen run volumes significantly; additionally, theprotein content captured in each chamber was further fractionated via non-porousreversed-phase HPLC. The notion that isoelectric membrane-based devices could

Fig. 2. (a) Exploded view of the miniaturized multi-compartment electrolyzer operating with

isoelectric membranes. An assembly with only 5 chambers is shown (3 sample chambers and the

two termini electrodic reservoirs). (b) Schematic diagram of the MCE for initial plasma frac-

tionation. The four upper disks represent the isoelectric membranes to be sandwiched in between

each chamber (by courtesy of Proteome Systems).


not capture very high pI proteins has been recently misspelled by Lalwani et al.(2004a). These authors used high pI membranes fabricated with quaternaryammonium derivatives of cyclodextrins and poly(vinyl alcohol), cross-linked withglycerol-1,3-diglycidyl ether and demonstrated compatibility with catholytes ascaustic as 1 M sodium hydroxide. By the same token, this same group (Lalwaniet al., 2004b) has produced hydrolytically stable, low-pI isoelectric membranes fromlow-pI ampholytic components, poly(vinyl alcohol), and a bifunctional cross-linker,glycerol-1,3-diglycidyl ether. The low-pI ampholytic components used contain oneamino group and at least two weakly acidic functional groups. These new, very low-pI isoelectric membranes have been successfully used as anodic membranes in iso-electric trapping separations with pH<1.5 anolytes and have been found to be agood replacement for the hydrolytically less stable polyacrylamide-based isoelectricmembranes. Now the circle is closed and no one can any longer claim that IPGscannot capture very low and very high pI proteins!

Perhaps, though, one of the limiting steps in fractionating samples with thesedevices is the length of time needed to capture a given protein population in a givenchamber, due to the sieving properties of the isoelectric membranes. A remedy to the

Fig. 3. Coomassie brilliant blue stained 2-D gels of the alkaline MCE fraction from a membrane

preparation of log phase yeast. The alkaline fraction was separated in the first dimension IPG

using 2% ASB 14 detergent. The excess detergent has combined with SDS to form mixed micelles

in the second dimension gel and caused the smearing observed in the low molecular mass part of

the gel. The 2-D gel is the display of a 1.0 mg membrane protein preparation using an 11 cm pH

7�10.5 IPG for the first dimension and GelChipTM 8�18% T second dimension gel. The gel is

annotated with 237 proteins, representing 93 unique gene products (from Pedersen et al., 2003, by

permission).


slow migration of proteins in MCEs due to the sieving effect of isoelectric mem-branes has been recently proposed by Cretich et al. (2003) who suggested usinghydrogel beads, in lieu of membranes, as pI barriers sandwiched in between thevarious chambers. Although this approach greatly reduced the focusing time, it wasplagued by the presence of electroendoosmotic flow (EOF), as the beads did notprovide a flow-tight system. Aware of that, we designed new amphoteric beads,composed of ionic acrylamide derivative monomers co-polymerized within the poresof a central ceramic hard core, minimizing thus mass transfer resistance of proteinsthat are transiently adsorbed onto the beads (Fortis et al., 2005a). Additionally,these beads exhibit a much reduced EOF, thus permitting fast separations in MCEdevices (Fortis et al., 2005b) with minimal liquid flux from chamber to chamber. It isanticipated that isoelectric beads will find a role in proteomic applications as a resultof a rapid separation in miniaturized devices.

An interesting variant to the use of the MCE apparatus could be a kind of direct2D method, as depicted in Fig. 4, by which the surface charge fractionation, asobtained in the various MCE fractions, is coupled to size discrimination by runningdirectly the content of each MCE chamber into an SDS gel. The bands eluted fromthe latter step would then be analyzed directly by MS (Cottingham, 2003).

MINIATURIZED ISOELECTRIC SEPARATION DEVICES

Although in the previous section we have mentioned ‘‘miniaturized’’ multi-chamber instruments, in reality these approaches still accommodate sizable samplevolumes in each chamber, of the order of 0.5�2 ml. Smaller devices have beenrecently described. In one approach Tan et al. (2002) have built a device consisting in96 mini-chambers (~75 ll each) arranged in eight rows. Neighbouring chambers in agiven row are separated by short glass tubes (4 mm innerdiameter, ID, 3 mm long),within which isoelectric hydrogels of specific pH values are polymerized. Duringfocusing, the device is sandwiched between blocks incorporating reservoirs forcatholyte and anolyte. This device has been used not for the fractionation of pro-teins, but rather of their digests. With the described set up, however, some reser-vations are to be underlined; one of them is that peptides will collect mostly aroundacidic and basic pI values, leaving preciously little few (mostly those containing Hisresidues) in the pH 5�8 range. In the 2-DE map displayed in Fig. 2a of Tan et al.(2002) the peptides deriving from the digest of four protein markers are visiblygrouped into vertical pillars centered around the following pIs: 4.5, 6.0, 8.8 and 10.0.In another approach, Zilberstein et al. (2003) proposed parallel processing in iso-electric focusing chips. The main separation tool, here, is a dielectric membrane(chip) with conducting channels that are filled by isoelectric hydrogels of varying pHvalues. The membranes are held perpendicularly to the applied electric field andproteins are trapped in the channels whose pH values are equal to the pI of theproteins. Further progress in these parallel, miniaturized devices has been reportedby the same group (Zilberstein et al., 2004a, 2004b).

In yet a third approach, a system called ‘‘Off-gel IEF’’ has been described byRos et al. (2002). Just like the multi-compartment separation technique, the systemhas been devised for the separation of proteins according to their pI and for theirdirect recovery in solution without adding buffers or ampholytes. The principle is to


place a sample in a liquid chamber which is positioned on top of an IPG gel.Theoretical calculations and modelling have shown that the protonation of anampholyte occurs in the thin layer of solvation closed to the IPG gel/solutioninterface (Arnaud et al., 2002). Upon application of a voltage gradient, perpendic-ularly to the liquid chamber, the electric field penetrates into the channel and extractsall charged species (those having pI values above and below the pH of the IPG gel),thus vacating them from the sample cup. After separation, only the globally neutralspecies (pI = pH of the IPG gel) remain in solution. In a further extension of thisinitial work, the system was improved and adapted to a multi-well device, composed

Fig. 4. Schematic diagram of a 2-D method interfacing MCE fractions with SDS-PAGE.

Upper drawing: MCE instrument assembled with seven sample and two electrodic chambers

(the numbers on top refer to the pIs of the various isoelectric membranes). Lower drawing:

loading of the content of each chamber directly into SDS-PAGE gels. The bands resolved

after the second step will then be analysed by MS (Courtesy of D. Speicher, Winstar

Institute).


of a series of compartments of small volume (100�300 ll) and compatible withcurrent instruments for separation (Michel et al., 2003) (see Fig. 5).

PRE-FRACTIONATION ON SEPHADEX BEDS

Contrary to all above-described methods Gorg et al. (2002), reported a tech-nique of sample pre-fractionation with neutral beads of dextran (Sephadex) to isolateproteins by isoelectric focusing prior to 2-DE map analysis. This is in fact a re-discovery of the well known ‘‘Radola (1973, 1975) technique’’, described in the 70s.When the method was first introduced it became quite popular and as a consequencecommercial products were designed to ‘‘guillotine’’ out the entire Sephadex cake into20 pieces after migration (for a more thorough description of the principle and theset-up, see Righetti, 1983). The focusing process is of course induced by the presenceof carrier ampholytes, the Sephadex beads being exploited only as an anticonvectivemedium. Proteomic analysis that follows is not often compatible with the presence ofcarrier ampholytes, which are difficult to remove. Mass spectrometry is, for instance,not compatible, since also carrier ampholytes would be detected and not easilydistinguished from peptides of similar molecular masses.

ACKNOWLEDGMENTS

PGR is supported by FIRB 2001 (No. RBNE01KJHT), PRIN 2005 (MURST,Rome), Fondazione Cassa di Risparmio di Verona (Bando 2002) and by the

Fig. 5. Experimental set-up used to perform Off-Gel separations with multi-cup devices, com-

posed of either 10 (a) or 22 (b and c) wells in different pH intervals, as specified under the gels

(from Michel et al., 2003, by permission).


European Community (proposal No. 12793, Allergy Card, 2005). GC is supportedby TELETHON (GP0019Y01), the ‘‘Foundation for Renal Disease in Children’’and FFC#13/2003 ‘‘Proteomics of the airway surface liquid: implication for cysticfibrosis’’.

REFERENCES

Anderson, L. N. and Anderson, N. G. (2002) Mol. Cell. Proteomics 1:845�867.Arnaud, I. L., Josserand, J., Rossier, J. S., and Girault, H. H. (2002) Electrophoresis 23:3253�3261.Bae, S. H., Harris, A. G., Hains, P. G., Chen, H., Garfin, D. E., Hazell, S. L., Paik, Y. K., Walsh, B., and

Cordwell, S. J. (2003) Proteomics 3:569�579.Bier, M. (1998) Electrophoresis 19:1057�1063.Bier, M., Mosher, R. A., Thormann, W., and Graham, A. (1984) In: Electrophoresis ’83 (H. Hirai, ed.),

de Gruyter, Berlin, pp. 99�107.Bordini, E., Hamdan, M., and Righetti, P. G. (2000) Rapid Commun. Mass Spectrom. 14:840�848.Burggraf, D., Weber, G., and Lottspeich, F. (1995) Electrophoresis 16:1010�1015.Campostrini, N., Areces, L. B., Rappsilber, J., Pietrogrande, M. C., Dondi, F., Pastorino, F., Ponzoni, M.,

and Righetti, P. G. (2005) Proteomics 5: 2385�2395.Chiari, M., Righetti, P. G., Negri, A., Ceciliani, F., and Ronchi, S. (1992) Electrophoresis 13:882�884.Cordwell, S. J., Nouwens, A. S., Verrills, N. M., Basseal, D. J., and Walsh, B. J. (2000) Electrophoresis

21:1094�1103.Corthals, G. L., Molloy, M. P., Herbert, B. R., Williams, K. L., and Gooley, A. A. (1997) Electrophoresis

18:317�323.Corthals, G. L., Wasinger, C. V., Hochstrasser, D. F., and Sanchez, J. C. (2000) Electrophoresis

21:1104�1115.Cottingham, K. (2003) J. Proteome Res. 2:574�574.Cretich, M., Pirri, G., Carrea, G., and Chiari, M. (2003) Electrophoresis 24:577�581.Davidsson, P., Paulson, L., Hesse, C., Blennow, K., and Nilsson, C. L. (2001) Proteomics 1:444�452.Dreger, M. (2003) Eur. J. Biochem. 270:589�599.Egen, N. B., Bliss, M., Mayersohn, M., Owens, S. M., Arnold, L., and Bier, M. (1988) Anal. Biochem.

172:488�494.Fortis, F., Girot, P., Brieau, O., Boschetti, E., Castagna, A., and Righetti, P. G. (2005a) Proteomics 5:

620�628.Fortis, F., Girot, P., Brieau, O., Castagna, A., Righetti, P. G., and Boschetti, E. (2005b) Proteomics 5:

629�638.Fountoulakis, M. and Juranville, J. F. (2003) Anal. Biochem. 313:267�282.Gorg, A., Boguth, G., Kopf, A., Reil, G., Parlar, H., and Weiss, W. (2002) Proteomics 2:1652�1657.Gygi, S. P., Corthals, G. L., Zhang, Y., Rochon, Y., and Aebersold, R. (2000) Proc. Natl. Acad. Sci. USA

97:9390�9395.Hannig, K. (1967) In: Electrophoresis: Theory, Methods and Applications, Vol. 2 (M. Bier, ed.), Academic

Press, New York, pp. 423�471.Hannig, K. (1982) Electrophoresis 3:235�242Hayakawa, M., Hosogi, Y., Takiguchi, H., Shiroza, T., Shibata, Y., Hiratsuka, K., Kiyama-Kishikawa,

M., Hamajima, S., and Abiko, Y. (2003) Anal. Biochem. 313:60�67.Herbert, B. R. and Righetti, P. G. (2000) Electrophoresis 21:3639�3648.Herbert, B., Pedersen, S. K., Harry, J. L., Sebastian, L., Grinyer, J., Traini, M. D., McCarthy, J. T.,

Wilkins, M. R., Gooley, A. A., Righetti, P. G., Packer, N. H., and Williams, K. L. (2003) Phar-

maGenomics 3:22�36.Herbert, B., Righetti, P. G., McCarthy, J., Grinyer, J., Castagna, A., Laver, M., Durack, M., Rummery,

G., Harcourt, R. and Williams, K. L. (2004) In: Purifying Proteins for Proteomics (R. J. Simpson,

ed.), Cold Spring Harbor Lab. Press, Cold Spring Harbor, pp. 431�442.Hjerten, S. (1967) Chromatogr. Rev. 9:122�219.Hochstrasser, A. C., James, R. W., Pometta, D., and Hochstrasser, D. (1991) Appl. Theor. Electrophor.

1:333�337.


Hoffmann, P., Ji, H., Moritz, R. L., Connolly, L. M., Frecklington, D. F., Layton, M. J., Eddes, J. S., and

Simpson, R. J. (2001) Proteomics 1:807�818.Horvath, Z. S., Gooley, A. A., Wrigley, C. W., Margolis, J., and Williams, K. L. (1996) Electrophoresis

17:224�226.Hoving, S., Voshol, H., and van Oostrum, J. (2000) Electrophoresis 21:2617�2621.Huber, L. A., Pfaller, K., and Vietor, I. (2003) Circulation Res. 92:962�968.Ivory, C. F. (2004) Electrophoresis 25:360�374.Issaq, H. J., Conrads, T. P., Janini, G. M., and Veenstra, T. D. (2002) Electrophoresis 23:3048�3061.Jonsson, M. and Rilbe, H. (1980) Electrophoresis 1:3�14.Jung, E., Heller, M., Sanchez, J. C., and Hochstrasser, D. F. (2000) Electrophoresis 21:3369�3377.Kobayashi, H., Shimamura, K., Akaida, T., Sakano, E., Tajima, N., Funazaki, J., Suzuki, H., and

Shinohara, E. (2003) J. Chromatogr. A 990:169�178.Kuhn, R. and Wagner, H. (1989) J. Chromatogr. 481:343�350.Lalwani, S., Shave, E., Fleisher, H. C., Nzeadibe, K., Busby, M. B., and Vigh, G. (2004a) Electrophoresis

25:2128�2138.Lalwani, S., Shave, E., and Vigh, G. (2004b) Electrophoresis 25:3323�3330.Lescuyer, P., Hochstrasser, D. F., and Sanchez, J. -C. (2004) Electrophoresis 25:1125�1135.Locke, V. L., Gibson, T. S., Thomas, T. M., Corthals, G. L., and Rylatt, D. B. (2002) Proteomics

2:1254�1260.Lopez, M. F. (2000) Electrophoresis 21:1082�1093.Margolis, J., Corthals, G., and Horvath, Z. S. (1995) Electrophoresis 16:98�100.Michel, P. E., Reymond, P., Arnaud, I. L., Josserand, J., Girault, H. H., and Rossier, J. S. (2003)

Electrophoresis 24:3�11.Pedersen, S. K., Harry, J. L., Sebastian, L., Baker, J., Traini, M. D., McCarthy, J. T., Manoharan, A.,

Wilkins, M. R., Gooley, A. A., Righetti, P. G., Packer, N. H., Williams, K. L., and Herbert, B. (2003)

J. Proteome Res. 2:303�312.Pieper, R., Gatlin, C. L., Makusky, A. J., Russo, P. S., Schatz, C. R., Miller, S. S., Su, Q., McGrath, A.

M., Estock, M. A., Parmar, P. P., Zhao, M., Huang, S. T., Zhou, J., Wang, F., Esquer-Blasco, R.,

Anderson, N. L., Taylor, J., and Steiner, S. (2003) Proteomics 3:1345�1364.Pietrogrande, M. C., Marchetti, N., Dondi, F., and Righetti, P. G. (2002) Electrophoresis 23:283�291.Pietrogrande, M. C., Marchetti, N., Dondi, F., and Righetti, P. G. (2003) Electrophoresis 24:217�224.Rammensee, H.G. (2004) Nature 427:203�204.Righetti, P.G. (1983) Isoelectric Focusing: Theory, Methodology and Applications. Elsevier, Amsterdam

129�136.Righetti, P. G., Wenisch, E., and Faupel, M. (1989) J. Chromatogr. 475:293�309.Righetti, P. G., Wenisch, E., Jungbauer, A., Katinger, H., and Faupel., M. (1990) J. Chromatogr.

500:681�696.Righetti, P. G. (1990) Immobilized pH Gradients: Theory and Methodology. Elsevier, Amsterdam 1�400.Righetti, P. G., Faupel, M. and Wenisch, E. (1992) In: Advances in Electrophoresis, Vol. 5 (A. Chrambach,

M. J. Dunn and B. J. Radola, eds.), VCH, Weinheim, pp. 159�200.Righetti, P. G., Castagna, A., and Herbert, B. (2001) Anal. Chem. 73:320A�326A.

Righetti, P. G., Castagna, A., Herbert, B., Reymond, F., and Rossier, J. S. (2003a) Proteomics

3:1397�1407.Righetti, P. G., Castagna, A., and Hamdan, M. (2003b) In: Advances in Chromatography, Vol. 42 (P. R.

Brown and E. Grushka, eds.), M. Dekker, New York, pp. 270�321.Ros, A., Faupel, M., Mees, H., Oostrum, J. V., Ferrigno, R., Reymond, F., Michel, P., Rossier, J. S., and

Girault, H. H. (2002) Proteomics 2:151�156.Shang, T. Q., Ginter, J. M., Johnston, M. V., Larsen, B. S., and McEwen, C. N. (2003) Electrophoresis

24:2359�2368.Southan, C. (2004) Proteomics 4:1712�1726.Stannard, C., Brown, L. R., and Godovac-Zimmermann, J. (2004) Curr. Proteomics 1:13�25.Tan, A., Pashkova, A., Zang, L., Foret, F., and Karger, B. L. (2002) Electrophoresis 23:3599�3607.Tracy, N. I. and Ivory, C. F. (2004) Electrophoresis 25:1748�1757.Wall, D. B., Kachman, M. T., Gong, S., Hinderer, R., Parus, S., Misek, D. E., Hanash, S. M., and

Lubman, D. M. (2000) Anal. Chem. 72:1099�1111.


Wang, H., Kachman, M. T., Schwartz, D. R., Cho, K. R., and Lubman, D. M. (2002) Electrophoresis

23:3168�3181.Weber, G., Islinger, M., Weber, P., Eckerskorn, C., and Volkl, A. (2004) Electrophoresis 25:1735�1747.Wenger, P., de Zuanni, M., Javet, P., Gelfi, C., and Righetti, P. G. (1987) J. Biochem. Biophys. Methods

14:29�43.Westbrook, J. A., Yan, J. X., Wait, R., Welson, S. Y., and Dunn, M. J. (2001) Electrophoresis

22:2865�2871.Wildgruber, R., Harder, A., Obermeier, C., Boguth, G., Weiss, W., Fey, S. J., Larsen, P. M., and Goerg,

A. (2000) Electrophoresis 21:2610�2616.Xiao, Z., Conrads, T. P., Lucas, D. A., Janini, G. M., Schaefer, C. F., Buetow, K. H., Issaq, H. J., and

Veenstra, T. D. (2004) Electrophoresis 25:128�133.Zhu, K., Yan, F., O’Neil, K. A., Hamler, R., Lin, L., Barder, T. J., and Lubman, D. M. (2003) Curr. Prot.

Protein Sci. :2331�23328.Zhu, Y. and Lubman, D. M. (2004) Electrophoresis 25:949�958.Zilberstein, G. V., Baskin, E. M., and Bukshpan, S. (2003) Electrophoresis 24:3735�3744.Zilberstein, G. V., Baskin, E. M., Bukshpan, S., and Korol, L. E. (2004a) Electrophoresis 25:3643�3651.Zilberstein, G. V., Korol, L. E., Bukshpan, S., and Baskin, E. M. (2004b) Proteomics 4:2533�2540.Zischka, H., Weber, G., Weber, P. J. A., Posch, A., Braun, R. J., Buhringer, D., Schneider, U., Nissum,

M., Meitinger, T., Ueffing, M., and Eckerskorn, C. (2003) Proteomics 3:906�916.Zuo, X. and Speicher, D. W. (2002) Proteomics 2:58�68.


How to Bring the “Unseen” Proteome to the Limelight via Electrophoretic PreFractionation...

Documents

Transcript of How to Bring the “Unseen” Proteome to the Limelight via Electrophoretic PreFractionation...