peer reviewed: prefractionation techniques in proteome analysis

$: Peer Reviewed: Prefractionation Techniques in Proteome Analysis$
3 2 0 A A N A LY T I C A L C H E M I S T R Y / J U N E 1 , 2 0 0 1

A new approach identifies morelow-abundance proteins.

Pier Giorgio Righetti and Annalisa CastagnaUniversity of Verona (Italy)

Ben HerbertProteome Systems (Australia)

PrefractionationTechniques

inProteome Analysis

J U N E 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y 3 2 1 A

roteomics is an emerging

area of research in the postgenomic

era that deals with the global analy-

sis of gene expression via a combination

of techniques for resolving, identifying,

quantitating, and characterizing pro-

teins. Bioinformatics is a fundamental

part of proteomics for storing and com-

municating data. Thus, proteomic and

genomic data can be linked with bioin-

formatics for mapping information

from genome projects (1). Although

proteomics and genomics can be ap-

plied independently, their impact can

be maximized when used in concert to

study complex biological problems.


For the past 25 years, two-dimen-sional polyacrylamide gel electrophore-sis (2-D PAGE) has been the techniqueof choice for analyzing the protein com-position of a given cell type and for mon-itoring changes in gene activity through thequantitative and qualitative analysis of thethousands of proteins that orchestrate various cellu-lar functions. Proteins are usually the functional molecules and,therefore, the most likely components to reflect qualitative (ex-pression of new proteins, post-translational modifications) andquantitative (up and down regulation, coordinated expression)differences in gene expression. Notwithstanding its extraordinaryresolving power, even 2-D PAGE has plateaued as to the num-ber of proteins that can be resolved and detected in a single map.The advent of immobilized pH gradients (IPGs) (2) has certain-ly made a big improvement, but the technique’s resolving powercannot be pushed any further. Solubilizing cocktails have alsobeen improved by adding new surfactants (3) and reducingagents (4); however, this progress seems to be leveling off.Staining protocols have not produced any increase in detectionsensitivity in the past five years (5). With an estimated 30,000genes in the human genome, and as many as 1 million proteinsin the proteome, improvements in resolving power and detec-tion sensitivity for tracking trace components is imperative.

Some recent papers suggest that the protein detection prob-lem could be overcome by running a series of narrow-range (<1pH unit) IPG strips on large-size gels (>18 cm in the first di-mension; large-format slabs in the second dimension) to dra-matically increase resolution (6). The entire wide-range 2-D mapwould then be electronically reconstructed with the narrow-range maps. This technique may give unreliable results, becauseeven very narrow IPG strips have to be loaded with the entire celllysate, which contains proteins focusing all along the pH scale.Aggregation among unlike proteins will cause massive precipita-tion. Additionally, the proteins that focus in the chosen narrow-range IPG interval will be strongly underrepresented becausethey will be only a small fraction of the entire sample loaded.

The gravity of this problem has been debated in a recent workby Gygi et al. (7). These authors analyzed a yeast lysate by load-ing 0.5 mg total protein on a narrow-range IPG at pH 4.9–5.7.Although they could visualize ~1500 spots by silvering, a largenumber of polypeptides simply did not appear in such a 2-Dmap. In particular, proteins from genes with codon bias values of<0.1 (low-abundance proteins) were not found, even thoughfully one-half of all yeast genes fall into that range. (The codonbias value for a gene is its propensity to use only one of severalcodons to incorporate a specific amino acid into the polypeptidechain.) Highly expressed proteins have large codon bias values(>0.2); thus, the authors concluded that only generally abundantproteins (codon bias >0.2) can be properly identified when ana-lyzing protein spots from 2-D maps by MS. The number of spotson a 2-D gel is not representative of the overall number or class-es of expressed genes that can be analyzed.

Loading only 40 µg total yeast lysate—as done in the early daysof 2-D mapping—Gygi et al. have calculated that only polypep-

pl4.5 9.5

(a) EFP SODBTPX

CRR GREA

FRRNUSG

RBFARPLI FABZ

G1788641

CSPC

GLNK

ECOFUR

NDK

CSPGCSPA

HNSMOPBBCP

RPLL

TRXA YJGF

(b) MALM YJGK

GRXCBOLA

ACCBYFHF GRXC

FLGE (fragment)FLGM

YCAR CSPG YGIN YRBA

(c) TPX YRDC YQGECSPA

YGADYRDA

YJGF

CSPA

NDKCSPC

(d) SODA

TPX

YGAD YJGF

CSPA

NDK

PSPE

G1788971

YHBH CSPE

G1788146

PPIA

CSPC

(e) HISAEFP

G1788136 TPX

APT

SODA SODB

YAJQG1788841

NDK

YFHF YFID

YJGFCSPATRXA G1788358 (fragment)

PABB (fragment)

YGBB

YFFB

YBGC

G1786688

YCGL

SODA

Figure 1. Partial 2-D gel images of the (a) input and the pools (b) 2,(c) 4, (d) 5, and (e) 0 of fractions collected from an hydroxyapatite pre-fractionation of a total E. coli cell lysate, which demonstrates the en-richment of lowMr proteins. The proteins were eluted with progres-sively increasing salt gradients of (b) 50 mM MgCl2, (c) 1.5 M MgCl2 ,(d) 1.5 M NaCl, and (e) 2.5 M NaCl. The samples were analyzed on apH 3–10 nonlinear IPG strip, followed by 9–16% T gradient SDS-PAGEgel. The gels were stained with colloidal Coomassie Blue, destainedwith water, and scanned. The proteins were identified by MALDI-TOFMS. The abbreviated names or numbers next to the protein spots arethose of the E. coli database (ftp://ncbi.nlm.nih.gov/genbank/genomes/bacteria/Ecoli/ecoli.ptt) (Adapted with permission from Ref. 15.)


tides with an abundance of at least 51,000 copies/cell could be de-tected (7). With only 0.5 mg, proteins present at 1000 copies/cellcan now be visualized by silvering; but those present at 10 and 100copies/cell can not. They concluded that the large range of pro-tein expression levels limits the ability of the 2-D MS approach toanalyze proteins of medium to low abundance, so the potential ofthis technique for proteome analysis is likewise limited. This is a se-vere limitation, as the portion of proteome presently missing isquite likely needed by researchers to understand cellular and regu-latory proteins. Such low-abundance polypeptide chains are typi-cally regulatory proteins. Thus, isoelectric focusing (IEF)/IPG-sodium dodecyl sulfate (SDS)-PAGE, coupled to MS or MS/MS,does not appear to be suitable for the global detection of proteinsin each cell. Moreover, the construction of complete, quantitativeprotein maps based on the MS approach will be very challenging,even for relatively simple, unicellular organisms.

Prefractionation can be a way out of this impasse. Chromato-graphic and electrophoretic methods are the two current ap-proaches to prefractionation.

DDiiffffeerreenntt cchhrroommaattooggrraapphhiicc aapppprrooaacchheessFountoulakis’ group extensively developed the following ap-proach. In their first procedure, they adopted affinity chroma -tography on heparin gels as a prefractionation step for enrichingcertain protein fractions in the bacterium Haemophilus in flu -enzae (8, 9). This Gram-negative bacterium is of pharmaceuticalinterest. Its complete genome comprises ~1740 open readingframes, although not more than 260 proteins have been char-acterized by 2-D map analysis (10). Heparin is a highly sulfatedglucosaminoglycan with affinity for a broad range of proteinssuch as nucleic acid-binding proteins and growth and protein syn-thesis factors. Because of its sulfate groups, heparin also func-tions as a high-capacity cation exchanger. Thus, prefractionationon heparin gels was deemed suitable for enriching low-copy-number gene products. In fact, ~160 cytosolic proteins boundwith different affinities to the heparin matrix were thus highlyenriched prior to 2-D PAGE separation. As a result, >110 newprotein spots detected in the heparin fraction were identified.

In their second approach (11), the same lysate of H.influenzae was prefractionated by chromatofo-

cusing on ion-exchange columns. Two pro-teins in the eluent, major ferric iron-bindingprotein (HI0097) and 5´-nucleotidase(HI0206), were obtained in a pureform. Another potential eluent protein(HI0052) was purified to near homo-geneity. Four other proteins, aspartateammonia lyase (HI0534), peptidaseD (HI0675), elongation factor Ts(HI0914), and 5-methyltetra hydro -pteroyl tri gluta mate methyltrans-ferase (HI1702), were strongly en-riched by the chromatofocusingprocess. Approximately 125 proteinswere identified in the eluent collectedfrom the column. Seventy were iden-

tified for the first time after this chromofocusing step, and mostwere low-abundance enzymes with various functions. Thus,with this additional step, 300 proteins were identified for H.influenzae by 2-D map analysis from ~600 spots.

In another approach, the cytosolic soluble proteins of H. in-fluenzae were prefractionated by Fountoulakis et al. using hy-drophobic interaction chromatography (HIC) on a phenyl col-umn (12). The eluent was subsequently analyzed by 2-Dmapping, and the spot characterized by MALDI time-of-flightMS (MALDI-TOF MS.) Approximately 150 proteins bound tothe column were identified, but only 30 were previously un-known. In addition, major spots represented most of the proteinsenriched by HIC, so the low-copy-number gene products wereonly modestly enriched. The chromatographic techniques identi-fied a total of 502 proteins (13). The same heparin chroma -tography procedure was subsequently applied by Karlsson et al. tothe prefractionation of human fetal brain soluble proteins (14).Approximately 300 proteins were analyzed, representing 70 dif-ferent polypeptides, 50 of which were bound to the heparin ma-trix. Eighteen brain proteins were identified for the first time. Thepolypeptides enriched by heparin chromatography included bothminor and major components of the brain extract. The enrichedproteins belonged to several classes, including proteasome com-ponents, dihydro pyrimidinase-related proteins, T-complex pro-tein 1 components, and enzymes with various catalytic activities.

Using another approach, Fountoulakis et al. reportedly en-riched low-abundance E. coli proteins by hydroxyapatite chro-matography (15). The complete genome of E. coli has now been

Pump

ReservoirsStirrers

Electrolyzer

Figure 2. Schematic drawing of the multicompartment electrolyzeroperating with isoelectric membranes. The holding platform isshown; flasks, power supply, and connecting tubings are not shown.

sequenced, and its proteome has been analyzed by 2-D mapping.To date, 223 unique loci have been identified and 201 protein en-tries were found on the SWISS-PROT 2-D PAGE on the ExPASyserver using the sequence retrieval system query tool (http://www.expasy.ch/www/sitemap.html). Of the 4289 possible geneproducts of E. coli, ~1200 spots could be counted on a typical 2-D map when ~2 mg total protein were applied. Most of the re-maining proteins may not have been expressed in sufficientamounts to be visualized following staining with Coom assie Blue,necessitating a prefractionation step on hydroxyapatite beads. Bythis procedure, ~800 spots, corresponding to 296 different pro-teins, were identified in the hydroxyapatite eluent. Approximately130 new proteins not detected in 2-D gels of the total extract wereidentified. This chromatographic step enriched the low-abundanceproteins and the major com-ponents of the E. coli extract.In particular, it enriched manylow molecular mass (Mr) pro-teins such as cold-shock pro-teins (CSPs).

Figure 1 is a series of pan-els of partial 2-D gel imagesof unfractionated sample andfractions collected from thehydroxy apatite prefractiona-tion of E. coli total lysate,demonstrating an efficient enrichment of many low Mr CSPssuch as CSP-G, CSP-A, CSP-C, and CSP-E.

Although the work presented by Fountoulakis’ group is im-pressive and truly innovative, this approach has some inherentdrawbacks. In general, huge concentrations of salts (>2.5 MNaCl) are needed for elution from chromatographic columns.As a consequence, some proteins could be lost during manipu-lations such as dialysis for salt removal and dilute fraction con-centration. Moreover, the eluted fractions do not representnarrow pI cuts; however, they generally are constituted by pro-teins with pIs in the pH 3–10 range. Therefore, wide pH gra-dients should be used for analysis.

A high salt concentration can induce irreversible adsorption ofproteins to the fractionation resin, resulting in irreversible loss ofspots during the subsequent 2-D mapping. Moreover, wheneluting from ion-exchange columns, the collected fractions willbe quite dilute, require a concentration step, and contain hugeamounts of salt, rendering them incompatible withthe IEF/IPG first-dimension step. Thus, al-though the approach by Fountoulakiscould be quite attractive for analyzingsome protein fractions in a total celllysate, it might not work for explor-ing the entire proteome.

EElleeccttrroopphhoorreettiicc sstteeppssOne possible approach describedby Hochstrasser et al. (16) is to ob-tain narrow pI cuts by using a mul-tichamber device developed by

Bier’s group (17). This equipment consists of a cooling fin-ger with a rotating cell that is divided into 20 compartmentsby nylon net. The protein mixture is present in the cell,which rotates slowly on its axis to prevent electrodecantation.At the end of the separation process, all of the chambers aresimultaneously emptied with the help of 20 syringe needlesconnected to a vacuum port. This apparatus is based on con-ventional IEF protocols; that is, it uses soluble carrier ampholytesfor driving a focusing process. As such, it is subject to all the prob-lems of conventional IEF—irreproducibility from run to run be-cause of synthetic buffer batch variability, cathodic drift, and pro-tein precipitation in the overloaded chambers. In addition, becausethe various chambers are divided only by a simple nylon net andare not “flow tight”, there is no drift control between chambers.

Another approach—usinga fractionation machine—isalso based on two flows. Aliquid curtain flowing down-stream in a narrow gap be-tween two flat glass platescoupled to an orthogonalelectric field separates macro-molecules as they are passive-ly transported into 80 collec-tion tubes. Burggraf et al.used this approach to pre-

fractionate complex sample mixtures (18).A multifunctional electrokinetic membrane apparatus that can

process and purify protein solutions based on differences of mo-bility, pI, and size is also a potential approach (19, 20). AlthoughCorthals et al. used this approach for prefractionation before 2-Danalysis, it is doubtful that high resolution was achieved. This in-strument can typically only use membranes based on size frac-tionation, and it provides only crude resolution (21).

MMuullttiiccoommppaarrttmmeenntt eelleeccttrroollyyzzeerrssThis kind of equipment represents an evolutionary step in allpreparative electrokinetic fractionation processes. The original ap-paratus devised by our group (Figure 2) consists of a stack ofchambers sandwiched between an anodic and a cathodic reservoir(22–24). The apparatus is modular and can accommodate up toeight flow chambers—six for sample collection and two as elec-trodic reservoirs. A multichannel peristaltic pump connects flowchambers to corresponding reservoirs, which are positioned out-

side the electric field. The reservoirs act as heat sinks and puri-fy larger sample volumes than could possibly be accom-

modated inside the flow chambers (typically restrictedto a total of 5 mL). The apparatus is thus based ontwo orthogonal flows: a hydraulic flow that trans-ports all sample components into and out of theelectric field until steady-state conditions arefound for all species; and an electric flow that al-lows each protein in the system to reach thechamber, where it will find isoelectric conditions.

This system is based on a new selectivity prin-ciple in separation science—permeability through

Because only protein cofo-

cusing in the same IPG inter-

val will be present, much

higher sample loads are fea-


a barrier via surface-charge modulation.Two isoelectric membranes facing eachflow chamber act by continuously titrat-ing the protein of interest to its pI. Theisoelectric membranes are highly selec-tive, retaining any proteins with pIs be-

tween their limiting values and allowingtransmigration of any nonamphoteric, non-

isoelectric species. The only condition requiredis pIcm > pIp > pIam, where the subscripts cm and

am denote cathodic and anodic membranes, respective-ly, and p is the protein with an isoelectric point between the twomembranes. For this mechanism to be operative, it is necessarythat the two isoelectric membranes possess good buffering capac-ity (�) to effectively titrate the protein present in the flow chamberto its pI while ensuring good current flow through the system.

Wenger et al. synthesized amphoteric, isoelectric membranesand demonstrated their use as good buffers at their respectivepIs (25). These membranes are created with the same technol-ogy used to make IPG strips. As such, they generate a single pHvalue, which will be the pI value of each membrane produced.The concept of isoelectric membranes is quite revolutionary.They can be envisioned as pH-dictating assemblies in an IEF sep-aration, much like the control provided by a pH-stat unit duringa biochemical reaction or an in vitro tissue growth experiment.

Each species that is tangent or crosses such isoelectric mem-branes is titrated to the pH of the membrane if it does not over-come its intrinsic � value. For amphoteric compounds, this resultsin a drastic change in mobility, which could reach zero if the twomembranes delimiting a single flow chamber have pIsencompassing the protein of interest. Other am-photeric species with lower or higher pI val-ues will be forced to exit from such achamber either toward the anode orcathode, respectively. With the properset of membranes, researchers can de-fine isoelectric conditions for a singlecomponent of a protein mixture andultimately arrest it as the sole isoelec-tric species. If the protein concentra-tion is high enough, the macro ionpresent in the liquid stream will pos-sess enough buffering power to controlthe pH if exogenous ions do not migratethrough the system.

Although this system was used originally for ex-treme purification of a single protein form for crystallization orbiochemical assays, it was logical to extend it to proteome analysis.Instead of using very narrow pI gaps between two membranes,large pI gaps (up to several pH units) can be used to trap groupsof proteins in a chamber. Such narrow-pI range families could beanalyzed on an IPG strip encompassing just the pI values.

The advantages of this procedure are immediately apparent. Itis fully compatible with the subsequent first-dimension separation.Such a prefractionation protocol is based on the same concept ofimmobilized pH gradients, and thus protein mixtures harvested

from the various chambers of this apparatus can be loaded ontoIPG strips without further treatment. They are isoelectric and de-void of any nonamphoteric ionic contaminant. The protocol per-mits casting isoelectric membranes with the same high precisioninherent in IPG technology, namely achieving pH reproducibilityto the second decimal place. It allows harvesting a population ofproteins with pI values precisely matching the pH gradient of anyIPG strip. When an entire cell lysate is analyzed in a wide gradient,protein precipitation is less likely. On the other hand, when thesame mixture is analyzed in a narrow gradient, massive precipita-

tion of all nonisoelectric proteins can occur with a strong riskof coprecipitation of proteins that would otherwise

focus in the narrow pH interval. Because only theproteins cofocusing in the same IPG interval will

be present, much higher sample loads are fea-sible, permitting detection of low-abundanceproteins. Finally, in samples containing ex-treme ranges of protein concentrations—such as human serum, in which a single pro-tein, albumin, represents >60% of the totalspecies—an isoelectric trap narrow enoughto just eliminate the unwanted protein could

be assembled. This also permits much highersample loads without interference from the most

abundant species.This technology was recently applied to sample pre-

fractionation in preparation for 2-D map analysis (26). How-ever, the apparatus in Figure 2 was too complex for proteomeanalysis, requiring as much as 25 ml of sample solution per cham-ber when operated in the recycling mode. Thus, the equipmenthad to be miniaturized to render it more suitable for 2-D map-ping. Ideally, the electrolyzer would hold only the total liquid vol-ume that could be adsorbed in the IPG strip for the first-dimen-sion run. In properly made strips, such a sample volume could beas much as 1 mL.

Figure 3 shows two 2-D maps in the pH 4–5 interval. One wasobtained with this prefractionation protocol (Figure 3b), whereas

100

70

50

30

20

10

FFIIGGUURREE 33.. (a) Silver stained 2-D map of an E. coli entire cell lysate,run in a 18 cm pH 4–5 gradient in the first dimension (300 mg sampleload) and (b) colloidal Coomassie G250 stained gel of the sample pre-fractionated with a miniaturized multicompartment electrolyzer).(Adapted with permission from Ref. 26.)



the other is available on the Internet at the SWISS-2-D-PAGE site(Figure 3a). Although the gel in Figure 3b is stained withCoomassie Brilliant Blue, more spots are visible on the silver-stained SWISS-2-D-PAGE E. coli map (Figure 3a). Moreover, be-cause the gel in Figure 3b is stained with Coommassie BrilliantBlue and not with silver, almost all of the spots are present in suf-ficient quantities for MS analysis. Although this is a clear indicationthat the prefractionation procedure should not lose componentsvia trapping into the isoelectric membranes, a protein assay on thestarting and ending products confirmed a 95% protein recovery(26). A similar assay performed on the ground and extracted iso-electric membranes failed to reveal any appreciable amount of pro-teinaceous material bound or adsorbed onto those surfaces.

Human serum provides an interesting example of the power ofthis prefractionation technique. Once albumin is trapped in a suit-able chamber (pI 5.6–6.1), higher sample loads can be applied

throughout the pH 3–10 interval. For example, inthe basic region, we applied a high-enough

sample load to reveal >230 spots, an in-credible number. All yielded good

quality spectra by MALDI-TOFMS, and 130 polypeptides wereidentified via searches of cur-rently available databases.The remaining 100 spotsmight represent novelpolypeptides of possible di-agnostic value. At present,this alkaline region in seraand in many 2-D maps of tis-sue extracts is devoid of spots.

These maps are quite similar tothose drawn by cartographers in

the 16th century, where big,empty areas were stamped hic sunt

leones (region populated by lions).The prefractionation method could prove

a formidable tool in proteome analysis, not only because it willprovide the much-needed improvement in resolution, but alsobecause of its increased sensitivity and its ability to load a muchhigher amount of sample in any narrow pH interval. The pro-tocol allows for such a high protein loading that onlyCoomassie stain will be needed for detection (24). Silver stain-ing is not suitable for MALDI-TOF procedures because thealdehydes used in most protocols generally cross-link proteinsand render them unavailable for further analysis. Finally, theglutaraldehyde-free silver stains commonly used for MS analy-sis are less sensitive and no better than colloidal Coomassie.

Supported, in part, by a grant from MURST (Coordinated Project 40%, Proteomeanalysis, 2000), ASI (Agenzia Spaziale Italiana, grant no. I/R/28/00), and theEuropean community (contract no. QLRT-2000-01903).

Pier Righetti is a professor of biochemistry at the University of Verona(Italy). His research interests include purification and crystallization,proteome analysis, and screening for genetic defects and DNA diagno-

sis. Annalisa Castagna is a postdoctoral fellow inRighetti’s laboratory. Her research interests arebiomedical applications of proteome analysis. BenHerbert is executive vice president of technologydevelopment at Proteome Systems (Australia). His re-search interests include novel methodologies in proteinanalysis. Address correspondence to Righetti at Dept. ofAgricultural and Industrial Biotechnologies, University of Verona,Strada Le Grazie No. 15, Verona 37134, Italy ([email protected]).

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peer reviewed: prefractionation techniques in proteome analysis

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