methods in shotgun proteome analysis

Trends Trends in Analytical Chemistry, Vol. 26, No. 1, 2007

Advances in chromatographictechniques and methods in shotgunproteome analysisMingliang Ye, Xiaogang Jiang, Shun Feng, Ruijun Tian, Hanfa Zou

Shotgun proteomics is a high-throughput approach to proteome analysis

whereby the protein mixture is digested and the peptides generated are

separated by capillary liquid chromatography and sequenced by tandem

mass spectrometry (MS). Due to the huge number of peptide species, sepa-

ration prior to MS analysis plays an important role in shotgun proteomics.

Overall sensitivity, dynamic range, throughput and general effectiveness of

shotgun proteomic analysis largely depend on how well the peptide mixture

is separated. In recent years, new separation techniques have been applied

successfully to proteome analysis and have dramatically improved protein

identification. We briefly review the recent development of chromato-

graphic techniques and methods in shotgun proteome analysis, including

the following three aspects: one-dimensional separation; multidimensional

separation; and, automated proteome-analysis systems.

ª 2006 Elsevier Ltd. All rights reserved.

Keywords: Automated proteome-analysis system; Multidimensional separation;

One-dimensional separation; Separation technique; Shotgun proteomics

Mingliang Ye, Xiaogang Jiang,

Shun Feng, Ruijun Tian,

Hanfa Zou*

National Chromatographic

R&A Center, Dalian

Institute of Chemical Physics,

Chinese Academy of Sciences,

Dalian 116023,

China

*Corresponding author.

Tel.: +86 411 84379610;

Fax: +86 411 84379620;

E-mail: [email protected]

80

1. Introduction

Proteomics aims to understand complexbiological systems by analyzing proteinexpression, protein function, protein mod-ifications and protein interactions. Massspectrometry (MS) is a central analyticaltechnique for proteome analysis. Accord-ing to the review paper published in Sciencein 2006 [1], there are in general four MS-based proteome-analysis strategies:(1) MS analysis of substantially purified

proteins;(2) MS analysis of complex peptide mix-

tures;(3) comparative pattern analysis; and,(4) hypothesis-driven strategies.

In all the above strategies, separationtechniques play an important role. Pro-teins or peptides in proteomic samplesneed to be separated prior to MS analysis.While the latter two approaches are stillin their infancy, the first two are widelyapplied in proteome research.

0165-9936/$ - see front matter ª 2006 Elsev

The first is exemplified by the classicproteomic approach: two-dimensional(2D) gel electrophoresis of proteinsfollowed by MS identification of proteins ingel spots. Although 2D gel electrophoresisprovides unprecedented separation powerfor proteins, this approach suffers severallimitations, including the difficulties ofresolving proteins with extreme size, pI orhydrophobicity, and the difficultiesassociated with automation and repro-ducibility.

In the second approach, also referred toas shotgun proteomics, complex proteinsamples are digested, the resultingpeptides are separated and then subject totandem MS (MS2) analysis, and the pro-teins are finally identified by searchingdatabases. Due to its good compatibilitywith on-line MS detection, reversed phaseliquid chromatography (RPLC) is typicallyused to separate peptides in shotgun pro-teomics as that circumvents some limita-tions of 2D gel electrophoresis. A majoradvantage of this approach is that a largenumber of proteins could be identified in ahigh-throughput manner.

Proteome analysis faces challengesbecause of the great complexity of proteinspecies and the large dynamic range ofprotein levels. In shotgun proteomics, thesamples are even more complex becausetryptic digestion generates several dozenpeptides per protein. For example, thereare 52,816 protein entries in Human IPIdatabase (version 2.23), and these pro-teins will generate 892,584 peptides aftertrypsin digestion in silico [2]. The numberof species is increased 17-fold. Due to thelimited scan rate of MS and ion-suppres-sion effects in MS, efficient separation prior

ier Ltd. All rights reserved. doi:10.1016/j.trac.2006.10.012

mailto:[email protected]

Trends in Analytical Chemistry, Vol. 26, No. 1, 2007 Trends

to MS is required. However, to separate such a complexsample is a big challenge in separation science.

In the past few years, some new separation techniqueswere developed for peptide separation and significantlyimproved the overall sensitivity, dynamic range,throughput and general effectiveness of shotgun pro-teomic analysis. We briefly review the contribution ofseparation techniques and methods to shotgun proteo-mics analysis, covering the following three aspects: one-dimensional separation; multidimensional separation;and, automated proteome-analysis systems.

2. One-dimensional separation

RPLC coupled on-line with electrospray MS2 is typicallyused for shotgun proteomic analysis because of the goodcompatibility of the mobile phase with MS detection. The75-lm · 12-cm capillary column packed with 5-lmporous C18 particles was often used for nanoflow RPLC-MS2analysis.

Although relatively complex mixtures can be sepa-rated well in RPLC, the analysis of mixtures in shotgunproteomic experiments containing thousands of peptides,which is extremely complex, has given rise to urgentdemand for development of an RP capillary column withhigher resolution. Reducing the diameter of chromato-graphic packing materials and increasing the columnlength are the most effective ways to achieve highlyefficient separations. To take advantage of long columnspacked with small particles, ultra-performance liquidchromatography (UPLC) has been developed by severalresearch groups [3–5]. Highly efficient analysis of BSAdigest by UPLC-MS2 was demonstrated by Tolley et al.[6] using a 22-cm · 150-lm column packed with1.5-lm C18 nonporous particles with applied pressuresvaried from 790 bar (11,500 psi) to 930 bar (13,500psi).

Shen et al. reported using an ultra-long column inUPLC for proteome analysis [7,8]. Fused silica capillarieswith length of 87 cm were packed with 3-lm C18 por-ous silica particles for separation of a proteolytic digest ofsoluble yeast proteins. Peak capacity up to about 1000was achieved at a back pressure of about 10,000 psi. LCefficiencies achieved with one-dimensional separation inUPLC provided good proteome coverage relative to theuse of moderate-efficiency LC involving two-dimensionalseparations. With improved separation, the co-elutedpeptides decrease, so the ionization suppression in MS isalleviated and that leads to improvement of detectionsensitivity.

Another effective way to improve detection sensitivityis to reduce the inner diameter of the separation columnas an electrospray mass spectrometer is a concentration-dependent detector. Proteome analysis using a narrow-i.d. separation column has been shown to increase the

detection sensitivity significantly [7–10]. For example,Haskins et al. [9] used a 25-lm i.d. capillary LC columnwith ion trap MS2 to enable identification of peptides atthe 60-amol level, and Shen et al. [10] used a 15-lm i.d.capillary LC column to enable identification and confir-mation of six tryptic peptides from only 7 amol of a BSAtryptic digest sample.

The use of ultra-long and ultra-narrow packed C18capillary columns offers the advantages of high peakcapacity, high detection sensitivity, and low sample andmobile-phase consumption. However, special high-pressure pumping equipment is required to operate thesesystems, and it is extremely difficult to pack capillarycolumns with i.d. less than 30 lm.

Monolithic columns have attracted a great deal ofinterest because of their ease of preparation, reliableperformance, good permeability and versatile surfacechemistry [11,12]. A 10-cm · 20-lm i.d. polystyrene-divinylbenzene monolithic column was reported to pro-vide 10-amol sensitivity demonstrated by the detectionof three peptides from a bovine catalase tryptic digest[13]. Although polymer-based monoliths have theadvantages of good biocompatibility and wide applica-tion range of pH values, they also undergo shrinking orswelling in organic solvents and may contain microporesthat adversely affect column efficiency and peak sym-metry. Silica-based monolithic columns have demon-strated high efficiencies and low back pressure as theirbimodal pore structures can be controlled independently(through pores and mesopores) [14,15].

Luo et al. [16] reported the preparation of extra-longoctadecylated silica-based monolithic capillary columnsfor proteome analysis. The monolithic column withdimension of 70 cm · 20 lm i.d. could be operated at amobile-phase pressure of 5000 psi providing a separa-tion peak capacity of �420 and the detection sensitivityof �15 amol. By connecting with a replaceable emitter,monolithic capillary columns can be readily interfacedwith electrospray ionization (ESI)-MS. Although using aseparate emitter is convenient and flexible, the con-nection of the tapered ESI emitter via a union willincrease extra-column volume and decrease separationefficiency. To circumvent this problem, we constructeda silica-based monolithic column with an integratedemitter [17]. Extremely sharp peaks were obtained forthe separation of tryptic digest of BSA, and that indi-cated the high efficiency of the integrated column. Thisgood performance was further demonstrated by using a60-cm monolithic capillary column to analyze yeastproteome as it resulted in the identification of 1323proteins.

Recently, Luo et al. [18] developed 10-lm i.d. silica-based monolithic capillary columns providing even moresensitive proteomics measurements. The nL/min flowrates from the 10-lm i.d. monolithic columns minimizecompound-to-compound variations in MS response and

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provide the basis for more quantitative measurementsfrom label-free analyses.

3. Multi-dimensional separation

Typical proteomic samples are too complex to achievesufficient fractionation using any of the current one-dimensional separation techniques, so various combi-nations of separation methods have been employed toobtain multi-dimensional peptide-separation systemswith sufficient separation power for comprehensiveproteome analysis. These include size-exclusion chro-matography followed by reversed-phase liquid chroma-tography (RPLC) [19], RPLC followed by capillaryelectrophoresis [20], strong cation-exchange chroma-tography (SCX) followed by RPLC [21–24], SCX followedby avidin affinity chromatography to select specificallybiotinylated peptides, and followed by RPLC [25,26], andisoelectric focusing (IEF) followed by RPLC [27–31].

Of these techniques, SCX followed by RPLC is mostwidely used. The SCX-RPLC steps can be either carriedout in tandem on-line [21,23] or with off-line fractioncollection between the dimensions [22,24,26]. The on-line mode has the advantages of low sample consump-tion and complete automation, while the off-line modehas the advantages of high resolution and high samplecapacity.

Using a biphasic column is a reliable, convenient wayto realize SCX-RPLC 2D separation [21,23,32]. The bi-phasic column was prepared by packing C18 and SCXparticles sequentially into a fused-silica nanospray tip.The acidified peptide mixture was bound onto the SCXsection. Discrete fractions of peptides were displaced fromthe SCX section directly onto the RP section, and thenseparated and eluted from the RP column into the massspectrometer. A major advantage of this separationtechnology is that the entire system is coupled directlyonline with MS, enabling a large number of peptides tobe directly identified in a high-throughput manner.

Even though SCX is effective in separating complexpeptide mixtures, it is not an ideal match with RPLC for2D separations because SCX separations partially dependon peptide hydrophobicity [24,33], so SCX is not com-pletely orthogonal to RPLC and the peak capacity of theSCX-RPLC combination is limited.

Unlike SCX, IEF bases the separation of peptides onlyon the peptide�s pI value, which is completely orthogonalto the hydrophobicity used in RPLC, so IEF is a goodalternative to SCX as the first dimension in a 2D sepa-ration system for shotgun proteomics. Using IEF forseparation of peptides has attracted a great deal ofinterest in recent years. Shen et al. [34] performed IEFseparations in fused-silica capillaries and found thatpeptides with pI-value differences as small as 0.01 couldbe resolved.

82 http://www.elsevier.com/locate/trac

Capillary IEF coupled online with capillary RPLC wasused for proteome analysis of yeast-cell lysate [29,30]. Itwas found that the overall peak capacity of this systemwas significantly improved over SCX-RPLC due to thecompletely orthogonal separation mechanisms of IEFand RPLC.

While high resolution has been achieved, the practicalapplication of capillary IEF in proteome research isseriously limited by the small loading capacity. Immo-bilized pH gradient (IPG) gels, typically used for IEF ofproteins, have also been applied for the fractionation ofpeptides as the first dimensional separation in shotgunproteomics [27,28]. IEF with IPG provides high-resolution separation, but its disadvantages are thetedious post-IEF sample processing that requires cuttingthe IPG gel strip into sections and extracting the peptidesfrom gel matrix.

In recent years, a series of solution-based IEF tech-niques, including a multi-chamber IEF device [31,33–35], off-gel electrophoresis [36,37] and free flowelectrophoresis [38,39], have been developed for theseparation of peptides. These techniques circumventedthe limitations of gel-based IEF and demonstrated thehigh resolution for the fractionation of peptides. Asignificant advantage of IEF over SCX for fractionation ofpeptides is that accurate peptide pI information could beobtained. It was reported that increased proteomecoverage and improved peptide identification confidencecould be achieved when the pI information was used tosort the results of database searching [27,37,38,40].

While multi-dimensional separations at the peptidelevel are very effective in solving the complexity problem,sample preparations at the protein level are more effectivein solving the dynamic range problem. Several high-abundance proteins in the proteome sample will generatehundreds of peptides after digestion, and that seriouslysuppresses the detection of peptides from low-abundanceproteins. In order to identify more low-abundance pro-teins by the shotgun proteomics approach, depletion ofthese high-abundance proteins before proteolysis ishighly recommended. To detect low-abundance proteinsin plasma or serum, a series of affinity-based approacheswas developed specifically to remove high-abundanceplasma proteins, such as albumin, immunoglobulins,antitrypsin, transferrin and haptoglobin [41,42]. Besidesaffinity chromatography, multi-dimensional chromato-graphic prefractionation at the protein level is also effec-tive in decreasing the interference of high-abundanceproteins with the detection of low-abundance proteins, asthese high-abundance proteins were only clustered in afew fractions after the fractionation. As an example, itwas reported that a total of 1292 distinct proteins wereidentified from plasma by prefractionation of the plasmaproteins by SCX-RP 2D chromatography followed withRPLC-MS2 analysis of the tryptic digest of each proteinfraction [43].

Trends in Analytical Chemistry, Vol. 26, No. 1, 2007 Trends

Instead of removing high-abundance proteins, an-other effective approach to detecting low-abundanceproteins is to enrich the low-abundance proteins. Forexample, affinity chromatography with immobilizedp-aminobenzamidine (ABA), an inhibitor of trypsin-likeserine proteases, was applied to enrich trypsin-like serineproteases in human plasma for shotgun proteomeanalysis [44].

4. Automated proteome-analysis system

To avoid labor-intensive operations and to obtain highlyreproducible results, it is necessary to automate the pro-teome-analysis system. Although the advantages ofnanoflow RPLC-MS2 are apparent for shotgun proteomeanalysis, automation of sample introduction onto theanalytical column remains a big challenge. In typicalcases, the proteomic sample size ranges from a fewmicroliter to 100 microliter. Due to the nanoliter (nL) flowrate adopted for separation of samples on the analyticalcolumn, it will take a long time if the sample is loadeddirectly onto the analytical column. To reduce the sample-loading time, a shorter, larger i.d. trap column maytherefore be used. A large-volume peptide sample is firstloaded onto the trap column at a fast rate in a short time,then the trapped peptides are eluted from the trap columnto a reversed phase analytical column. The automation ofthis system can easily be realized by directly connectingthe trap column with a switching valve [45–47]. How-ever, even using a nanoflow switching valve, the deadvolume attributed by the valve and the transfer lines is stillsignificant and degrades the performance of the chroma-tography separation. A specially vented column system,where trap column and analytical column were directlyconnected via a microcross with an open/close switchingvalve, has automated sample introduction [48,49]. Thedead volume significantly decreased due to the fact thatthe mobile phase for nanoflow RPLC separation does notpass through the switching valve.

While a C18 trap column has been used in mostautomated sample-introduction systems, it was foundrecently that separation performance could be improvedwhen an SCX trap column was used [50]. Because thepeptide sample is retained on the entrance end of theseparation C18 capillary column before the gradientstarted for separation, the dead volume before the cap-illary column hardly affected the separation adversely.The SCX trap column system was demonstrated to be agood substitute for the system using C18 trap column forautomation of nanoflow RPLC-MS2.

The use of a biphasic column is the simplest way toautomate SCX-RP 2D separations for large-scale prote-ome analysis [32]. The system requires only onequaternary pump. Two channels of the pump generate abinary organic solvent (water/acetonitrile) gradient for

RP chromatography and another two channels generatea salt step gradient for SCX chromatography. Use of avolatile buffer permits the high salt solution to be flushedthrough the capillary biphasic column, which is coupleddirectly with MS. While the 2D separation of the systemis automated, manual sample introduction is required,and that is labor intensive.

The SCX trap-column system developed to automatesample introduction for nanoflow RPLC- MS2 can also beperformed in 2D for large-scale analysis [50], which isfully automated, including sample introduction. Theautomation of SCX-RP 2D separations can also beachieved by column switching using separate pumpsystems for providing salt gradient [51]. The advantagesof the column-switching approach are rapid sampleloading, injection of large-volume samples and nointroduction of salt into the mass spectrometer. Fur-thermore, column switching allows the independentcontrol and optimization of the two dimensions.

Besides separation, the automated proteome-analysissystem should include sample-processing steps, such assample clean-up and protein digestion. Using theimmobilized enzyme reactor is a good way to realize theautomation of protein digestion. It was reported thattrypsin-based monolithic reactor with dimension of50-mm · 4.6-mm i.d. was coupled on-line with anLC-MS2 system for automated protein digestion andprotein identification [52]. However, the large size of thereactor is not suitable for use in a nanoflow LC-MS2

system. Recently, nL-scale monolithic microreactorswere developed for rapid digestion of minute samples[53,54]. The microreactor coupled on-line with thenanoflow LC-MS2 system allowed fast proteome analysisof a minute, complex protein sample.

5. Conclusion

In recent years, a series of new separation techniqueshas been applied successfully to proteome analysis andhas provided dramatic improvement in protein identifi-cation, although complete coverage of proteins for anyorganism has not yet been accomplished. Analyticalchallenges remain in trying to resolve the problems ofcomplexity and dynamic range. Besides the developmentof high-resolution separation techniques, the develop-ment of information-rich separation techniques, wherephysical properties of the peptides, such as pI values andhydrophobicity, could be obtained precisely, is prefera-ble, as these properties could be used to sort the results ofdatabase searching to increase the confidence in proteinidentifications and to improve the coverage of proteomes.

New specific approaches to remove high-abundanceproteins and new approaches to enrich low-abundanceproteins efficiently need to be developed to increase fur-ther the ability to detect more low-abundance proteins in

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complex proteome samples. A fully automated proteome-analysis system integrated with sample processing, pep-tide separation and detection, and data processing needto be established and optimized to improve thethroughput of shotgun proteome analysis.

Acknowledgements

This work was supported by National Natural SciencesFoundation of China (No. 20327002, 20675081), theChina State Key Basic Research Program Grant(2005CB522701), and the Knowledge Innovation pro-gram of DICP to H.Z. and National Natural SciencesFoundation of China (No. 20605022) to M.Y. aregratefully acknowledged.

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