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Quantitative proteomics using mass spectrometry
70
Quantitative proteomics using mass spectrometry Salvatore Sechi and Yoshiya Oday
The use of stable isotopes as internal standards in mass spectrometry has opened a new era for quantitative proteomics. Depending on the point at which the label is introduced, most procedures can be classi®ed as in vivo labeling, in vitro pre-digestion labeling or in vitro post-digestion labeling. In vivo labeling has been used for cells that can be grown in culture and has the advantage of being more accurate. The pre-digestion and post-digestion labeling procedures are suitable for all types of sample including human body ¯uids and biopsies. Several new mass spectrometric strategies mark signi®cant achievements in determining relative protein concentrations and in quantifying post-translational modi®cations. However, further technology developments are needed for understanding the complexity of a dynamic system like the proteome. Addresses Correspondence: National Institute of Diabetes and Digestive and Kidney Diseases, NIH, DHHS, Bethesda, MD 20892-5460, USA e-mail: [email protected] y Eisai Co., Ltd, Laboratory of Seeds Finding Technology, Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan
Current Opinion in Chemical Biology 2003, 7:70±77 This review comes from a themed issue on Proteomics and genomics Edited by Matthew Bogyo and James Hurley 1367-5931/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1367-5931(02)00010-8 Abbreviations 2DE two-dimensional gel electrophoresis FT-ICR Fourier transform ion cyclotron resonance ICAT isotope-coded af®nity tag MALDI matrix-assisted laser desorption ionization MCAT mass-coded abundance tagging
Introduction
The complete sequences of more than 100 genomes and the nearly complete sequences of many others, including the human (http://www.nlm.nih.gov), are giving a new vision to the study of biological systems. However, function is usually ful®lled from the proteins containing the actual information rather than the potential as indicated by the genes. Furthermore, gene expression varies with time and is different in different cell types within an organism; as a result, the proteome is by far more complex than the genome. Often, gene expression has been studied using mRNA arrays. However, in view of recent studies showing that there is no good correlation between mRNA quantities and protein quantities [1±3,4] we Current Opinion in Chemical Biology 2003, 7:70±77
ought to investigate biological problems also at the proteome level. It should be emphasized that post-translational modi®cations, regulation of protein function by proteolysis, and composition of macromolecular complexes can only be determined at the protein level. Most of the initial efforts in proteomics were focused on methods to effectively identify large numbers of proteins. Recent developments in mass spectrometry (MS) enable high-throughput identi®cation of proteins. However, to understand the function of a protein, changes in gene expression often have to be determined, thus quantitative protein pro®ling is an essential part of proteomics. For this purpose, several technologies to identify and quantify proteins from biological samples have been developed. Two-dimensional gel electrophoresis (2DE) in combination with MS has been used for many years for studying the proteome (see [5] for a recent review). The development of a two-colour ¯uorescent labeling system, which allows two protein samples (e.g. control and disease) to be differentially labeled and analysed in the same gel, obviates many of the reproducibility problems inherent to 2DE (see [6] for a review). Even using this system, however, quantitative results might sometimes be ambiguous because of the presence of more than one protein in a single spot. Moreover, 2DE is labour intensive and dif®cult to automate. Protein array technology has been recently developed and used for identifying novel ligands and for quantitative pro®ling (see [7] for a review). Although this approach has been used successfully, keeping proteins in their native and active conformations is still a challenge. MS has played a major role in quantitative proteomics, and here we review the recent developments.
The role of stable isotopes for quantitation
In quantitative proteomics, the main sources of error are variations in the sample preparation procedures (e.g. protein extraction, enrichment, fractionation) and/or variations in the analysis (in this case MS). These errors can be signi®cantly reduced using internal standards. An important characteristic of a standard is that its chemical±physical characteristics should be as similar as possible to the analyte to be analysed. For this reason, stable isotopes have been used since the early 1990s in peptide analysis [8±10]. However these early studies were limited to the quanti®cation of one or a few analytes and stable isotopes have only recently been used for quantitative proteomics. Normally, one sample is labeled with a heavy www.current-opinion.com
Quantitative proteomics using mass spectrometry Sechi and Oda 71
Figure 1
(a) In vivo labeling
State 1
(b) Pre-digestion in vitro labeling
(c) Post-digestion in vitro labeling
State 2 State 1
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State 2
State 1
Label Mix
Extract/Fractionate Digest
Extract Extract Fractionate Fractionate
Label
Extract Extract Fractionate Fractionate
Label Mix
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State 2
Enrich for Cys-peptides
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Relative quantitation from mass spectra Current Opinion in Chemical Biology
Schematic representation of quantitative proteomics procedures using MS. Depending on the point in the process where the label is introduced, most procedures can be classified as (a) in vivo labeling, (b) in vitro pre-digestion labeling or (c) in vitro post-digestion labeling. In the procedure that uses 18 O, the label is introduced during the digestion. However the two samples are mixed after the digestion, thus it could be categorized as post-digestion labeling.
reagent and a second sample is labeled with a light reagent. The two samples are then mixed and analysed by mass spectrometry. The ratio between the two isotopic distributions (one for the light reagent and one for the heavy reagent) can then be determined from the mass spectra and used to calculate the relative protein quantities. Several labeling approaches in combination with MS have been developed to do quantitative pro®ling. These methods can be mainly divided into three classes: in vivo labeling, in vitro pre-digestion labeling, and postdigestion labeling (Figure 1). In vivo labeling
The in vivo labeling method was ®rst described by Oda et al. [11]. In this procedure, yeast cells were grown in two separate media, one of which contained heavy isotopes (in this case 15 N). The two yeast cultures were combined, the proteins extracted, fractionated and then separated by gel electrophoresis. Finally, the proteins of interest were digested with trypsin before MS analysis and the relative quantities determined from the isotopic distribution ratios. In vivo labeling was later used in a similar way for analysing a whole cell lysate from bacteria [12,13,14]. www.current-opinion.com
In this study, the protein extracts from the whole cell lysate were digested with trypsin and the complex tryptic peptide mixture obtained was separated chromatographically. Fourier transform ion cyclotron resonance (FTICR) MS was then used to analyse and quantify the tryptic peptides. During the ®rst analysis, a series of accurate mass tags was generated and used as `biomarkers' for each speci®c protein. These tags were later used for high-throughput quantitation. Conrads et al. [15] also used FT-ICR-MS for the quantitative analysis of bacterial proteomes. To simplify the analysis, they puri®ed the cysteine-containing peptides from the complex tryptic peptide mixture using an af®nity tag that speci®cally binds to thiols. Recently, a method was developed that directly couples microscale two-dimensional chromatography to tandem mass spectrometry (MudPit) [16]. In this study, more than 800 proteins from mixtures of yeast cells, grown either in 14 N- or 15 N-enriched media, were identi®ed in a single experiment. This shows the potential of MudPit for high-throughput quantitative proteomics. Although these `controlled media' might in some cases be used for growing mammalian cells [16], their use would be limited and expensive. An alternative recently proposed Current Opinion in Chemical Biology 2003, 7:70±77
72 Proteomics and genomics
for studying mammalian cells is to utilize isotopically labeled leucine [17,18] or lysine [19]. In vivo labeling proved to be an effective way for performing quantitative proteomic experiments. However, this approach cannot be applied to tissues or body ¯uids and is limited to cells that can be grown in culture in `controlled media'. However, in in vivo labeling procedures, the internal standard is introduced early in the process, thus obviating the variations caused by sample preparation and giving higher accuracy to the quantitation. In vitro pre-digestion labeling
Gygi et al. [20] developed a quantitative proteome analysis using a class of reagents termed isotope-coded af®nity tags (ICATs) and electrospray ionization MS. For comparing the protein pro®les of yeast grown in two different conditions (galactose and ethanol as carbon source), they synthesized biotinylated iodoacetamide derivatives in a heavy form (deuterated) and in a light form, and used them to label the cysteines of the two protein extracts before combining and proteolyzing them with trypsin. The major innovation of this approach was that an af®nity tag (biotin) was used to purify cysteinecontaining peptides, reducing the complexity of a peptide mixture by about a factor of 10. As a result, several proteins that usually can't be observed in an approach like 2DE could be identi®ed and quanti®ed. Several studies have further proved the utility of these reagents in quantitative proteomics [21,22,23,24,25]. However, these ICAT reagents are relatively large and the presence in the MS/MS spectra of ions from the fragmentation of the af®nity label complicates the analysis. Recently, a commercial, improved, cleavable ICAT reagent was introduced (http://appliedbiosystems.com). This new reagent contains an acid-cleavable linker that allows the removal of the af®nity tag before MS analysis. By simplifying MS analysis, the number of proteins identi®ed and quanti®ed is increased (Y Oda et al., unpublished data). When evaluating the ICAT approach, the relatively high cost of the reagents and the several chromatographic steps involved before the MS analysis should also be considered. A simple proteomic approach using acrylamide and deuterated acrylamide for differentially labeling cysteines before electrophoresis separation was recently introduced [26,27]. This does not require any additional step to the typical peptide mapping method used for identifying proteins [27,28]. The identi®cation and quantitation were achieved on the same matrix-assisted laser desorption ionization (MALDI) spectra. Although this procedure is simple and the ICAT approaches have the power Current Opinion in Chemical Biology 2003, 7:70±77
of reducing the complexity of a protein mixture, both are limited to quantifying proteins that contain cysteines (about 8% of the proteins in yeast do not contain cysteines). In vitro post-digestion labeling
In several procedures, isotopic labels have been introduced after proteolysis. For example, Mirgorodskaya et al. [29] carried out the enzymatic digest in the presence of 18 O-water or regular water. The sample digested with 18 O-water incorporates 18 O, generating an isotopic label that was used for relative quantitation. This comprehensive approach for labeling all proteolytic peptides was used to compare the protein extracts from two serotypes of adenovirus [30]. However, the quantitation can be complicated by the possible loss or incomplete incorporation of the label. Another simple procedure for incorporating a label is to esterify the carboxyl groups of the tryptic peptides with deuterated methanol (CD3OH) [31]. This sample can then be mixed and compared with another sample that has been esteri®ed with regular methanol. One of the minor disadvantages of this approach is that the harsh esteri®cation condition requirement (hydrochloric acid solution) might produce partial deamidation of asparagine and glutamine residues, increasing the sample heterogeneity. In addition, this procedure should be carried out in anhydrous conditions, which can be dif®cult to obtain. The primary amine group is also a suitable target for introducing an isotopic label. The deuterated and nondeuterated forms of N-acetoxysuccinimide were used for differentially labeling the N-terminus and the e-amino group of lysines [32]. This approach was successfully applied to the quanti®cation of relatively simple protein mixtures [33,34]. It was later improved using an enrichment protocol for cysteine- and histidine-containing peptides [35] and combined with a C-terminus-labeling procedure [36]. The applicability of this protocol was further shown in a study performed using ion mobility MS [37]. In a strategy named mass-coded abundance tagging (MCAT), the e-amino group of lysine was also labeled [38]. Only one sample is modi®ed with the reagent (O-methylisourea) and compared with the unmodi®ed sample for determining the relative quantities. Although this procedure is simple and inexpensive, several issues related to the difference in the chemical±physical characteristics between the labeled and unlabeled peptides markedly reduce the accuracy of the quantitation. Following the concept of enrichment introduced with the ICAT reagent, Zhou et al. [39] developed a solid-phase isotope-tagging reagent with a photocleavable linker that binds to cysteine-containing peptides. Using this reagent, the enrichment of the cysteine-containing peptides and www.current-opinion.com
Quantitative proteomics using mass spectrometry Sechi and Oda 73
the incorporation of the isotopic label are achieved in a single step, making the whole process much simpler.
Quantitation of post-translational modi®cation level of proteins
Many biological processes are regulated by post-translational modi®cations. Phosphorylation and glycosylation are amongst the most common modi®cations and MS has been successfully used for their identi®cation. In many cases, however, phosphorylation is a dynamic process with complex kinetics involving several amino acids within a single protein (for examples see http:// www.lecb.ncifcrf.gov/phosphoDB/). Some of the in vitro pre-digestion and post-digestion labeling procedures described in this review are not suitable for the quantitation of phosphorylation because they isolate speci®c peptides (e.g. the ICAT procedure isolates cysteine-containing peptides). On the contrary, a procedure such as in vivo labeling is suitable for this kind of study and it was used for the identi®cation and quanti®cation of several sites of the protein Ste20 [11]. Labeled synthetic stan-
dards were also used for the quanti®cation of several phosphorylation sites [40,41]. However, the requirement of synthesizing a labeled peptide for each site investigated limits this approach to a few focus studies. To investigate the phosphoproteome or a large subset of the phosphoproteome, several enrichment procedures have been developed. In some cases, these procedures involved speci®c chemical reaction methods for the phosphate group [42±44,45], metal af®nity column [46], or antibodies [47,48]. Perhaps the most extensive characterization of the phosphoproteome was done by Ficarro et al. [46], who characterized more than 1000 phosphopeptides. Although this study was limited to the identi®cation of phosphorylation sites, the approach seems extendable to quantitation. Figure 2 describes the general strategies for the quanti®cation of phosphorylation sites. A main difference between the various strategies is that when using an enrichment method, only changes in phosphorylation
Figure 2
(a)
Label phosphopeptides
(b)
In vivo uniform labeling
Pi
Pi
Pi
Pi
State 1
State 2
State 1
State 2
Mix/digest
Mix/digest
Digest of selected proteins Enrich for phosphopeptides
Pi Pi
Pi
Pi
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∗ m/z m/z
: Phosphorylated peptide
peptide ∗ : Unphosphorylated from same protein Current Opinion in Chemical Biology
Schematic representation of the strategies for quantitative phosphoproteomics. (a) In some strategies the phosphopeptides are labeled and these labels are used for affinity purification. (b) In other cases, the in vivo labeling procedure is used and all peptides in the digest are labeled. When the enrichment procedure is applied, only phosphopeptides are detected and quantified (left bar chart), while the in vivo labeling approach has the potential of quantifying all peptides in the digest (right bar chart). www.current-opinion.com
Current Opinion in Chemical Biology 2003, 7:70±77
74 Proteomics and genomics
Table 1 Summary of pros and cons for quantitative proteomics using MS. Method
Pros
Cons
In vivo labeling
Simple and accurate Labeling of all peptides is achieved Reduced complexity by enrichment of cysteine-containing peptides Applicable to all types of sample Reduced complexity by enrichment of cysteine-containing peptides. Applicable to all types of sample Simple derivatization and procedure Applicable to all types of sample Easy derivatization Applicable to all types of sample Labeling of all peptides is achieved Easy derivatization Applicable to all types of sample Labeling of all peptides is achieved Applicable to all types of sample
Limited to cells that can be grown in culture
ICAT
Cleavable ICAT
Labeling with acrylamide Proteolysis in the presence of 16 O/18 O Esterification
Derivatization of primary amines MCAT Site-specific photo-cleavable method
Limited to the analysis of proteins containing cysteine Complex MS/MS spectra due to the presence of the affinity label Limited to the analysis of proteins containing cysteine Requires more steps than ICAT Limited to the quantification of proteins containing cysteine Complex analysis. Possible loss or incomplete incorporation of the label Derivatization efficiency might not be high
Applicable to all types of sample Inexpensive, no need for stable isotopes Reduced complexity by enrichment of cysteine-containing peptides Applicable to all types of sample
Requires multiple steps for derivatization increasing the possibility of errors and decreasing the accuracy Derivatization efficiency might not be high Requires multiple steps for derivatization Low accuracy Limited to the analysis of proteins containing cysteine
The most common methods used in quantitative proteomics are reported (left column). None of the above procedures works for all cases and they all have some pros (center column) or cons (right column) that are briefly described.
can be quanti®ed and the information about changes in gene expression are lost. These changes could be quanti®ed only by keeping in consideration the total protein (unmodified modified (e.g. phosphorylated)). On the other hand, an enrichment procedure is necessary for obtaining a better coverage of the phosphoproteome.
Conclusions
Current quantitative proteomics using MS is mainly based on the incorporation of stable isotope tags. Although numerous reports regarding protein quantitation using MS have been published, there are still many areas that require further developments (see Table 1 for a list of pros and cons). In vivo labeling procedures are potentially the most accurate because the label is introduced early in the process; however, it is limited to cells that can be grown in culture. The advantage of the pre-digestion and postdigestion labeling procedures is that they are suitable for all types of sample including human body ¯uids and biopsies. Proteomic technologies are developing at a very fast pace. Some recent efforts, mainly focusing on the identi®cation of proteins from Plasmodium falciparum [49,50], have reported major improvements in throughput. Perhaps the most comprehensive proteome characterization made to date is the identi®cation of 2528 proteins from rice [51]. Current Opinion in Chemical Biology 2003, 7:70±77
In several novel approaches, not described in depth in this review, stable isotopes were not used for the quantitation. For example, Chelius et al. [52] used the intensities in the ion chromatograms from two LC-MS spectra for determining the relative quantities and Stoeckly et al. [53] used the ion intensities from the MALDI spectra of tissue sections for generating semi-quantitative images. Protein chips together with mass spectrometry were also used for expression pro®ling (see [54] for a review). In this case, however, only pro®les without identifying the proteins responsible for the differences were obtained. The differences between samples were characterized as differences in pro®le shapes. Although the idea of obviating the use of stable isotopes is attractive, it seems that the alternatives still need further development before reaching the accuracy usually achieved with stable isotopes.
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10. Wang R, Sweeney D, Gandy SE, Sisodia SS: Detection and quanti®cation of amyloid beta protein and variants by immunoprecipitation-mass spectrometry. J Biol Chem 1996, 271:31894-31902. 11. Oda Y, Huang K, Cross FR, Cowburn D, Chait BT: Accurate quantitation of protein expression and site-speci®c phosphorylation. Proc Natl Acad Sci USA 1999, 96:6591-6596. In this study, the concept of using isotopically labeled media for a quantitative proteomic approach is introduced. The relative abundance of proteins present in the wild type versus mutant yeast cells is reported and the use of this approach for the quanti®cation of phosphorylation sites of the protein Ste20 is described. 12. Smith RD, Pasa-Tolic L, Lipton MS, Jensen PK, Anderson GA, Shen Y, Conrads TP, Udseth HR, Harkewicz R, Belov ME et al.: Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry. Electrophoresis 2001, 22:1652-1658. This study used isotopically enriched media for labeling the proteome of a microorganism and FT-ICR MS for the identi®cation and quanti®cation of the total cell lysate tryptic digest. The same approach was also used for the quanti®cation of intact proteins that were separated by capillary isoelectrofocusing before analysis by FT-ICR. 13. Smith RD, Anderson GA, Lipton MS, Pasa-Tolic L, Shen Y, Conrads TP, Veenstra TD, Udseth HR: An accurate mass tag strategy for quantitative and high-throughput proteome measurements. Proteomics 2002, 2:513-523. Deinococcus radiodurans cells were grown in isotopically enriched media for quantitation. For each identi®ed protein, an accurate mass tag AMT is generated by MS-MS (using an LCQ or FT-ICR instrument) and used for a later high-throughput identi®cation using a single-stage MS. In this study, several proteins were identi®ed and quanti®ed from the tryptic digest of the whole cell lysate. 14. Lipton MS, Pasa-Tolic' L, Anderson GA, Anderson DJ, Auberry DL, Battista JR, Daly MJ, Fredrickson J, Hixson KK, Kostandarithes H et al.: Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags. Proc Natl Acad Sci USA 2002, 99:11049-11054. 15. Conrads TP, Alving K, Veenstra TD, Belov ME, Anderson GA, Anderson DJ, Lipton MS, Pasa-Tolic L, Udseth HR, Chrisler WB et al.: Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine af®nity tags and 15N-metabolic labeling. Anal Chem 2001, 73:2132-2139. www.current-opinion.com
A novel approach that includes growing cells in 15 N-enriched media and labeling cysteines with an af®nity tag for puri®cation of cysteine-containing peptides is described. The cysteine-containing peptides were analysed by LC-FT-ICR MS. They showed that this approach can be used for bacterial cultures as well as mammalian cultures. 16. Washburn MP, Ulaszek R, Deciu C, Schieltz DM, Yates JR III: Analysis of quantitative proteomic data generated via multidimensional protein identi®cation technology. Anal Chem 2002, 74:1650-1657. In this study, a shotgun approach is used for the comparison of two yeast cultures grown in 14 N-minimal media and 15 N-enriched media. These two cultures were mixed and the proteins extracted from the whole cell lysate were digested with trypsin. This complex peptide mixture is ®rst separated by multidimensional LC and then analysed using an LCQ instrument. The identi®cation of more than 800 proteins in a single experiment indicates the great potential of this approach for high-throughput quantitative proteomics. 17. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M: Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 2002, 1:376-386. This study describes an alternative to the procedure of growing cells in 15 N-enriched media. The advantage of this approach is that the use of isotopically labeled leucine is more suitable to cells of mammalian origin. Although it seems to be of general use for culture cells it could not be applied to body ¯uids or tissues. 18. Jiang H, English AM: Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine. J Proteome Res 2002, 1:345. 19. Gu S, Pan S, Bradbury EM, Chen X: Use of deuterium-labeled lysine for ef®cient protein identi®cation and peptide de novo sequencing. Anal Chem 2002, 74:5774-5785. 20. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R: Quantitative analysis of complex protein mixtures using isotope-coded af®nity tags. Nat Biotechnol 1999, 17:994-999. 21. Grif®n TJ, Han DK, Gygi SP, Rist B, Lee H, Aebersold R, Parker KC: Toward a high-throughput approach to quantitative proteomic analysis: expression-dependent protein identi®cation by mass spectrometry. J Am Soc Mass Spectrom 2001, 12:1238-1246. 22. Han DK, Eng J, Zhou H, Aebersold R: Quantitative pro®ling of differentiation-induced microsomal proteins using isotopecoded af®nity tags and mass spectrometry. Nat Biotechnol 2001, 19:946-951. The approach of using ICAT reagents for protein quanti®cation and identi®cation is further extended. In this study, about 500 microsomal proteins are identi®ed and quanti®ed from human myeloid leukemia cells using a combination of ICAT and multidimensional chromatography separation before MS and MS-MS. 23. Grif®n TJ, Gygi SP, Rist B, Aebersold R, Loboda A, Jilkine A, Ens W, Standing KG: Quantitative proteomic analysis using a MALDI quadrupole time-of-¯ight mass spectrometer. Anal Chem 2001, 73:978-986. 24. Smolka M, Zhou H, Aebersold R: Quantitative protein pro®ling using two-dimensional gel electrophoresis, isotope-coded af®nity tag labeling, and mass spectrometry. Mol Cell Proteomics 2002, 1:19-29. This study shows that differential labeling with ICAT reagents can be successfully used in a 2D-PAGE experiment. However the af®nity puri®cation and therefore the af®nity tags are not needed in this kind of experiment and other, simpler procedures would be more suitable. 25. Arnott D, Kishiyama A, Luis EA, Ludlum SG, Marsters JC Jr, Stults JT: Selective detection of membrane proteins without antibodies: a mass spectrometric version of the western blot. Mol Cell Proteomics 2002, 1:148-156. 26. Sechi S, Chait BT: Modi®cation of cysteine residues by alkylation. A tool in peptide mapping and protein identi®cation. Anal Chem 1998, 70:5150-5158. 27. Sechi S: A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis. Rapid Commun Mass Spectrom 2002, 16:1416-1424. Current Opinion in Chemical Biology 2003, 7:70±77
76 Proteomics and genomics
This article further extends the use of deuterated acrylamide to quantitative proteomics. Two complex protein mixtures were separately labeled with acrylamide and deuterated acrylamide before electrophoresis. The ratios between the two isotopic distributions were used for reliably quanti®cation of several proteins present, in ratios from 1:1 to 1:10. 28. Gehanne S, Cecconi D, Carboni L, Righetti PG, Domenici E, Hamdan M: Quantitative analysis of two-dimensional gel-separated proteins using isotopically marked alkylating agents and matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2002, 16:1692-1698. This study con®rms that deuterated acrylamide can be used for quantitation of sample isolated by 2D-PAGE, as previously illustrated. 29. Mirgorodskaya OA, Kozmin YP, Titov MI, Korner R, Sonksen CP, Roepstorff P: Quantitation of peptides and proteins by matrixassisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards. Rapid Commun Mass Spectrom 2000, 14:1226-1232. This study describes the use of 18 O-water for relative quantitation of proteins. Issues relative to the overlap of the two isotopic distributions (one for peptides labeled with 18 O and one for the unlabeled peptides (16 O)), and the possibility of the incorporation of one or two 18 O during the digest, make the calculation of the relative quantities dif®cult. 30. Yao X, Freas A, Ramirez J, Demirev PA, Fenselau C: Proteolytic 18 O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Anal Chem 2001, 73:2836-2842. The use of 18 O-water previously described by Mirgorodskaya et al. [29] is further extended for the comparison of two adenovirus serotypes. Although the use of a high-resolution instrument like an FT-ICR solves the problems related to the overlap of the two isotopic distributions, it seems that the calculation of the relative quantities is still complex. 31. Goodlett DR, Keller A, Watts JD, Newitt R, Yi EC, Purvine S, Eng JK, von Haller P, Aebersold R, Kolker E: Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation. Rapid Commun Mass Spectrom 2001, 15:1214-1221. The relative quantities of proteins are determined by differential labeling of the tryptic digest of two samples with methanol and D3-methanol. This procedure of permethylation has the added bene®t of facilitating de novo sequencing. 32. Ji J, Chakraborty A, Geng M, Zhang X, Amini A, Bina M, Regnier F: Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J Chromatogr B Biomed Sci Appl 2000, 745:197-210. 33. Munchbach M, Quadroni M, Miotto G, James P: Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety. Anal Chem 2000, 72:4047-4057. Complex protein mixtures were separated by 2D- or 1D-PAGE and the spots to be identi®ed/quanti®ed were selected from two gels (e.g. treated and control). The proteins were succinilated with a heavy (D4) or light (D0) reagent before digestion with Asp/Glu-C. The conditions for succinilation of the lysine were improved and optimized to avoid secondary unwanted reactions. An added bene®t of this procedure is that the labeling of the Nterminus facilitates de novo sequencing because the b-ion intensities are greatly increased. Although this approach has several advantages, it does not overcome the issue of reproducibility that is inherent to 2DE. 34. Zhang R, Sioma CS, Wang S, Regnier FE: Fractionation of isotopically labeled peptides in quantitative proteomics. Anal Chem 2001, 73:5142-5149. In this study, the elution times between the heavy and the light forms of acetylated peptides are compared with the retention time of peptides modi®ed with ICAT reagents. Acetylated peptides co-elute independently from the label, whereas the light and heavy forms of the ICAT-modi®ed peptides have different retention times. 35. Wang S, Zhang X, Regnier FE: Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates. J Chromatogr A 2002, 949:153-162. The novelty of this procedure is that a puri®cation step for isolating cysteine- and/or histidine-containing peptides is used. The quanti®cation procedure is the same as previously described for succinic anhydride. The approach seems reasonable, although the copper af®nity column might not be exclusively selecting for histidine-containing peptides. 36. Liu P, Regnier FE: An isotope coding strategy for proteomics involving both amine and carboxyl group labeling. J Proteome Res 2002, 1:443. Current Opinion in Chemical Biology 2003, 7:70±77
37. Kindy JM, Taraszka JA, Regnier FE, Clemmer DE: Quantifying peptides in isotopically labeled protease digests by ion mobility/ time-of-¯ight mass spectrometry. Anal Chem 2002, 74:950-958. 38. Cagney G, Emili A: De novo peptide sequencing and quantitative pro®ling of complex protein mixtures using mass-coded abundance tagging. Nat Biotechnol 2002, 20:163-170. MCAT is introduced. Lysine guanidination, previously employed to facilitate sequencing by post-source decay (PSD), was used to facilitate sequencing by LC-MS/MS. The comparison of the isotopic distribution of unlabeled peptides with guanidinilated peptides was used for relative quantitation of yeast proteins. Although the average errors are within 25%, some peptides have errors as big as 62%, perhaps due to changes in ionization between lysine and guanidil-lysine. 39. Zhou H, Ranish JA, Watts JD, Aebersold R: Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat Biotechnol 2002, 20:512-515. This study describes a procedure for simultaneous solid-phase enrichment and labeling of cysteinyl peptides. The method described seems to be a signi®cant improvement to the typical ICAT approach. 40. Ruse CI, Willard B, Jin JP, Haas T, Kinter M, Bond M: Quantitative dynamics of site-speci®c protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry. Anal Chem 2002, 74:1658-1664. 41. Stemmann O, Zou H, Gerber SA, Gygi SP, Kirschner MW: Dual inhibition of sister chromatid separation at metaphase. Cell 2001, 107:715-726. In this article, labeled synthetic peptides were used as internal standards in an LC-MS experiment for quantifying the grade of phosphorylation of separase, a protein involved in chromatid separation during the anaphase. 42. Oda Y, Nagasu T, Chait BT: Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotechnol 2001, 19:379-382. 43. Adamczyk M, Gebler JC, Wu J: Selective analysis of phosphopeptides within a protein mixture by chemical modi®cation, reversible biotinylation and mass spectrometry. Rapid Commun Mass Spectro 2001, 15:1481-1488. 44. Zhou H, Watts JD, Aebersold R: A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 2001, 19:375-378. 45. Goshe MB, Veenstra TD, Panisko EA, Conrads TP, Angell NH, Smith RD: Phosphoprotein isotope-coded af®nity tags: application to the enrichment and identi®cation of lowabundance phosphoproteins. Anal Chem 2002, 74:607-616. Phospho Ser/Thr were labeled with EDT-D0 or EDT-D4 after b-elimination. The proof of principle is done using casein. In theory, this approach could be used for the quantitation of phosphorylation sites on a proteomic scale. However, using the described method, O-glycosylation sites would be also labeled and the procedure for avoiding the labeling of these sites has not been developed. 46. Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM: Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 2002, 20:301-305. This study describes a methodology for phosphoproteome characterization. Although it has not been used for quantitation purposes, this enrichment procedure is of general use for phosphopeptides. More than 1000 phosphopeptides were detected and 383 phosphorylation sites identi®ed in one experiment from yeast cell lysate. 47. Pandey A, Podtelejnikov AV, Blagoev B, Bustelo XR, Mann M, Lodish HF: Analysis of receptor signaling pathways by mass spectrometry: identi®cation of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc Natl Acad Sci USA 2000, 97:179-184. 48. Pandey A, Fernandez MM, Steen H, Blagoev B, Nielsen MM, Roche S, Mann M, Lodish HF: Identi®cation of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways. J Biol Chem 2000, 275:38633-38639. 49. Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL et al.: A proteomic view of the Plasmodium falciparum life cycle. Nature 2002, 419:520-526. www.current-opinion.com
Quantitative proteomics using mass spectrometry Sechi and Oda 77
50. Lasonder E, Ishihama Y, Andersen JS, Vermunt AM, Pain A, Sauerwein RW, Eling WM, Hall N, Waters AP, Stunnenberg HG, Mann M: Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 2002, 419:537-542. 51. Koller A, Washburn MP, Lange BM, Andon NL, Deciu C, Haynes PA, Hays L, Schieltz D, Ulaszek R, Wei J et al.: Proteomic survey of metabolic pathways in rice. Proc Natl Acad Sci USA 2002, 99:11969-11974. 52. Chelius D, Bondarenko PV: Quantitative pro®ling of proteins in complex mixtures using liquid chromatography and mass spectrometry. J Proteome Res 2002, 1:317. Determination of quantities base only on ion intensities from LC-MS spectra. This seems a preliminary but promising approach.
www.current-opinion.com
53. Stoeckli M, Chaurand P, Hallahan DE, Caprioli RM: Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nat Med 2001, 7:493. This article elegantly describes the use of MS for imaging. The image is generated by MALDI MS, scanning a tissue section imbedded with matrix. Several mouse brain sections are used to illustrate the technology. 54. Weinberger SR, Dalmasso EA, Fung ET: Current achievements using ProteinChip Array technology. Curr Opin Chem Biol 2002, 6:86-91. This article reviews the use of ProteinChip array for quantitation using surface enhanced laser-desorption (SELDI) protein chips.
Current Opinion in Chemical Biology 2003, 7:70±77
Quantitative proteomics using mass spectrometry Salvatore Sechi and Yoshiya Oday
The use of stable isotopes as internal standards in mass spectrometry has opened a new era for quantitative proteomics. Depending on the point at which the label is introduced, most procedures can be classi®ed as in vivo labeling, in vitro pre-digestion labeling or in vitro post-digestion labeling. In vivo labeling has been used for cells that can be grown in culture and has the advantage of being more accurate. The pre-digestion and post-digestion labeling procedures are suitable for all types of sample including human body ¯uids and biopsies. Several new mass spectrometric strategies mark signi®cant achievements in determining relative protein concentrations and in quantifying post-translational modi®cations. However, further technology developments are needed for understanding the complexity of a dynamic system like the proteome. Addresses Correspondence: National Institute of Diabetes and Digestive and Kidney Diseases, NIH, DHHS, Bethesda, MD 20892-5460, USA e-mail: [email protected] y Eisai Co., Ltd, Laboratory of Seeds Finding Technology, Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan
Current Opinion in Chemical Biology 2003, 7:70±77 This review comes from a themed issue on Proteomics and genomics Edited by Matthew Bogyo and James Hurley 1367-5931/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1367-5931(02)00010-8 Abbreviations 2DE two-dimensional gel electrophoresis FT-ICR Fourier transform ion cyclotron resonance ICAT isotope-coded af®nity tag MALDI matrix-assisted laser desorption ionization MCAT mass-coded abundance tagging
Introduction
The complete sequences of more than 100 genomes and the nearly complete sequences of many others, including the human (http://www.nlm.nih.gov), are giving a new vision to the study of biological systems. However, function is usually ful®lled from the proteins containing the actual information rather than the potential as indicated by the genes. Furthermore, gene expression varies with time and is different in different cell types within an organism; as a result, the proteome is by far more complex than the genome. Often, gene expression has been studied using mRNA arrays. However, in view of recent studies showing that there is no good correlation between mRNA quantities and protein quantities [1±3,4] we Current Opinion in Chemical Biology 2003, 7:70±77
ought to investigate biological problems also at the proteome level. It should be emphasized that post-translational modi®cations, regulation of protein function by proteolysis, and composition of macromolecular complexes can only be determined at the protein level. Most of the initial efforts in proteomics were focused on methods to effectively identify large numbers of proteins. Recent developments in mass spectrometry (MS) enable high-throughput identi®cation of proteins. However, to understand the function of a protein, changes in gene expression often have to be determined, thus quantitative protein pro®ling is an essential part of proteomics. For this purpose, several technologies to identify and quantify proteins from biological samples have been developed. Two-dimensional gel electrophoresis (2DE) in combination with MS has been used for many years for studying the proteome (see [5] for a recent review). The development of a two-colour ¯uorescent labeling system, which allows two protein samples (e.g. control and disease) to be differentially labeled and analysed in the same gel, obviates many of the reproducibility problems inherent to 2DE (see [6] for a review). Even using this system, however, quantitative results might sometimes be ambiguous because of the presence of more than one protein in a single spot. Moreover, 2DE is labour intensive and dif®cult to automate. Protein array technology has been recently developed and used for identifying novel ligands and for quantitative pro®ling (see [7] for a review). Although this approach has been used successfully, keeping proteins in their native and active conformations is still a challenge. MS has played a major role in quantitative proteomics, and here we review the recent developments.
The role of stable isotopes for quantitation
In quantitative proteomics, the main sources of error are variations in the sample preparation procedures (e.g. protein extraction, enrichment, fractionation) and/or variations in the analysis (in this case MS). These errors can be signi®cantly reduced using internal standards. An important characteristic of a standard is that its chemical±physical characteristics should be as similar as possible to the analyte to be analysed. For this reason, stable isotopes have been used since the early 1990s in peptide analysis [8±10]. However these early studies were limited to the quanti®cation of one or a few analytes and stable isotopes have only recently been used for quantitative proteomics. Normally, one sample is labeled with a heavy www.current-opinion.com
Quantitative proteomics using mass spectrometry Sechi and Oda 71
Figure 1
(a) In vivo labeling
State 1
(b) Pre-digestion in vitro labeling
(c) Post-digestion in vitro labeling
State 2 State 1
Label
State 2
State 1
Label Mix
Extract/Fractionate Digest
Extract Extract Fractionate Fractionate
Label
Extract Extract Fractionate Fractionate
Label Mix
Digest
State 2
Enrich for Cys-peptides
Digest
Digest
Label
Label Mix
Relative quantitation from mass spectra Current Opinion in Chemical Biology
Schematic representation of quantitative proteomics procedures using MS. Depending on the point in the process where the label is introduced, most procedures can be classified as (a) in vivo labeling, (b) in vitro pre-digestion labeling or (c) in vitro post-digestion labeling. In the procedure that uses 18 O, the label is introduced during the digestion. However the two samples are mixed after the digestion, thus it could be categorized as post-digestion labeling.
reagent and a second sample is labeled with a light reagent. The two samples are then mixed and analysed by mass spectrometry. The ratio between the two isotopic distributions (one for the light reagent and one for the heavy reagent) can then be determined from the mass spectra and used to calculate the relative protein quantities. Several labeling approaches in combination with MS have been developed to do quantitative pro®ling. These methods can be mainly divided into three classes: in vivo labeling, in vitro pre-digestion labeling, and postdigestion labeling (Figure 1). In vivo labeling
The in vivo labeling method was ®rst described by Oda et al. [11]. In this procedure, yeast cells were grown in two separate media, one of which contained heavy isotopes (in this case 15 N). The two yeast cultures were combined, the proteins extracted, fractionated and then separated by gel electrophoresis. Finally, the proteins of interest were digested with trypsin before MS analysis and the relative quantities determined from the isotopic distribution ratios. In vivo labeling was later used in a similar way for analysing a whole cell lysate from bacteria [12,13,14]. www.current-opinion.com
In this study, the protein extracts from the whole cell lysate were digested with trypsin and the complex tryptic peptide mixture obtained was separated chromatographically. Fourier transform ion cyclotron resonance (FTICR) MS was then used to analyse and quantify the tryptic peptides. During the ®rst analysis, a series of accurate mass tags was generated and used as `biomarkers' for each speci®c protein. These tags were later used for high-throughput quantitation. Conrads et al. [15] also used FT-ICR-MS for the quantitative analysis of bacterial proteomes. To simplify the analysis, they puri®ed the cysteine-containing peptides from the complex tryptic peptide mixture using an af®nity tag that speci®cally binds to thiols. Recently, a method was developed that directly couples microscale two-dimensional chromatography to tandem mass spectrometry (MudPit) [16]. In this study, more than 800 proteins from mixtures of yeast cells, grown either in 14 N- or 15 N-enriched media, were identi®ed in a single experiment. This shows the potential of MudPit for high-throughput quantitative proteomics. Although these `controlled media' might in some cases be used for growing mammalian cells [16], their use would be limited and expensive. An alternative recently proposed Current Opinion in Chemical Biology 2003, 7:70±77
72 Proteomics and genomics
for studying mammalian cells is to utilize isotopically labeled leucine [17,18] or lysine [19]. In vivo labeling proved to be an effective way for performing quantitative proteomic experiments. However, this approach cannot be applied to tissues or body ¯uids and is limited to cells that can be grown in culture in `controlled media'. However, in in vivo labeling procedures, the internal standard is introduced early in the process, thus obviating the variations caused by sample preparation and giving higher accuracy to the quantitation. In vitro pre-digestion labeling
Gygi et al. [20] developed a quantitative proteome analysis using a class of reagents termed isotope-coded af®nity tags (ICATs) and electrospray ionization MS. For comparing the protein pro®les of yeast grown in two different conditions (galactose and ethanol as carbon source), they synthesized biotinylated iodoacetamide derivatives in a heavy form (deuterated) and in a light form, and used them to label the cysteines of the two protein extracts before combining and proteolyzing them with trypsin. The major innovation of this approach was that an af®nity tag (biotin) was used to purify cysteinecontaining peptides, reducing the complexity of a peptide mixture by about a factor of 10. As a result, several proteins that usually can't be observed in an approach like 2DE could be identi®ed and quanti®ed. Several studies have further proved the utility of these reagents in quantitative proteomics [21,22,23,24,25]. However, these ICAT reagents are relatively large and the presence in the MS/MS spectra of ions from the fragmentation of the af®nity label complicates the analysis. Recently, a commercial, improved, cleavable ICAT reagent was introduced (http://appliedbiosystems.com). This new reagent contains an acid-cleavable linker that allows the removal of the af®nity tag before MS analysis. By simplifying MS analysis, the number of proteins identi®ed and quanti®ed is increased (Y Oda et al., unpublished data). When evaluating the ICAT approach, the relatively high cost of the reagents and the several chromatographic steps involved before the MS analysis should also be considered. A simple proteomic approach using acrylamide and deuterated acrylamide for differentially labeling cysteines before electrophoresis separation was recently introduced [26,27]. This does not require any additional step to the typical peptide mapping method used for identifying proteins [27,28]. The identi®cation and quantitation were achieved on the same matrix-assisted laser desorption ionization (MALDI) spectra. Although this procedure is simple and the ICAT approaches have the power Current Opinion in Chemical Biology 2003, 7:70±77
of reducing the complexity of a protein mixture, both are limited to quantifying proteins that contain cysteines (about 8% of the proteins in yeast do not contain cysteines). In vitro post-digestion labeling
In several procedures, isotopic labels have been introduced after proteolysis. For example, Mirgorodskaya et al. [29] carried out the enzymatic digest in the presence of 18 O-water or regular water. The sample digested with 18 O-water incorporates 18 O, generating an isotopic label that was used for relative quantitation. This comprehensive approach for labeling all proteolytic peptides was used to compare the protein extracts from two serotypes of adenovirus [30]. However, the quantitation can be complicated by the possible loss or incomplete incorporation of the label. Another simple procedure for incorporating a label is to esterify the carboxyl groups of the tryptic peptides with deuterated methanol (CD3OH) [31]. This sample can then be mixed and compared with another sample that has been esteri®ed with regular methanol. One of the minor disadvantages of this approach is that the harsh esteri®cation condition requirement (hydrochloric acid solution) might produce partial deamidation of asparagine and glutamine residues, increasing the sample heterogeneity. In addition, this procedure should be carried out in anhydrous conditions, which can be dif®cult to obtain. The primary amine group is also a suitable target for introducing an isotopic label. The deuterated and nondeuterated forms of N-acetoxysuccinimide were used for differentially labeling the N-terminus and the e-amino group of lysines [32]. This approach was successfully applied to the quanti®cation of relatively simple protein mixtures [33,34]. It was later improved using an enrichment protocol for cysteine- and histidine-containing peptides [35] and combined with a C-terminus-labeling procedure [36]. The applicability of this protocol was further shown in a study performed using ion mobility MS [37]. In a strategy named mass-coded abundance tagging (MCAT), the e-amino group of lysine was also labeled [38]. Only one sample is modi®ed with the reagent (O-methylisourea) and compared with the unmodi®ed sample for determining the relative quantities. Although this procedure is simple and inexpensive, several issues related to the difference in the chemical±physical characteristics between the labeled and unlabeled peptides markedly reduce the accuracy of the quantitation. Following the concept of enrichment introduced with the ICAT reagent, Zhou et al. [39] developed a solid-phase isotope-tagging reagent with a photocleavable linker that binds to cysteine-containing peptides. Using this reagent, the enrichment of the cysteine-containing peptides and www.current-opinion.com
Quantitative proteomics using mass spectrometry Sechi and Oda 73
the incorporation of the isotopic label are achieved in a single step, making the whole process much simpler.
Quantitation of post-translational modi®cation level of proteins
Many biological processes are regulated by post-translational modi®cations. Phosphorylation and glycosylation are amongst the most common modi®cations and MS has been successfully used for their identi®cation. In many cases, however, phosphorylation is a dynamic process with complex kinetics involving several amino acids within a single protein (for examples see http:// www.lecb.ncifcrf.gov/phosphoDB/). Some of the in vitro pre-digestion and post-digestion labeling procedures described in this review are not suitable for the quantitation of phosphorylation because they isolate speci®c peptides (e.g. the ICAT procedure isolates cysteine-containing peptides). On the contrary, a procedure such as in vivo labeling is suitable for this kind of study and it was used for the identi®cation and quanti®cation of several sites of the protein Ste20 [11]. Labeled synthetic stan-
dards were also used for the quanti®cation of several phosphorylation sites [40,41]. However, the requirement of synthesizing a labeled peptide for each site investigated limits this approach to a few focus studies. To investigate the phosphoproteome or a large subset of the phosphoproteome, several enrichment procedures have been developed. In some cases, these procedures involved speci®c chemical reaction methods for the phosphate group [42±44,45], metal af®nity column [46], or antibodies [47,48]. Perhaps the most extensive characterization of the phosphoproteome was done by Ficarro et al. [46], who characterized more than 1000 phosphopeptides. Although this study was limited to the identi®cation of phosphorylation sites, the approach seems extendable to quantitation. Figure 2 describes the general strategies for the quanti®cation of phosphorylation sites. A main difference between the various strategies is that when using an enrichment method, only changes in phosphorylation
Figure 2
(a)
Label phosphopeptides
(b)
In vivo uniform labeling
Pi
Pi
Pi
Pi
State 1
State 2
State 1
State 2
Mix/digest
Mix/digest
Digest of selected proteins Enrich for phosphopeptides
Pi Pi
Pi
Pi
Peptide mass fingerprint
∗ m/z m/z
: Phosphorylated peptide
peptide ∗ : Unphosphorylated from same protein Current Opinion in Chemical Biology
Schematic representation of the strategies for quantitative phosphoproteomics. (a) In some strategies the phosphopeptides are labeled and these labels are used for affinity purification. (b) In other cases, the in vivo labeling procedure is used and all peptides in the digest are labeled. When the enrichment procedure is applied, only phosphopeptides are detected and quantified (left bar chart), while the in vivo labeling approach has the potential of quantifying all peptides in the digest (right bar chart). www.current-opinion.com
Current Opinion in Chemical Biology 2003, 7:70±77
74 Proteomics and genomics
Table 1 Summary of pros and cons for quantitative proteomics using MS. Method
Pros
Cons
In vivo labeling
Simple and accurate Labeling of all peptides is achieved Reduced complexity by enrichment of cysteine-containing peptides Applicable to all types of sample Reduced complexity by enrichment of cysteine-containing peptides. Applicable to all types of sample Simple derivatization and procedure Applicable to all types of sample Easy derivatization Applicable to all types of sample Labeling of all peptides is achieved Easy derivatization Applicable to all types of sample Labeling of all peptides is achieved Applicable to all types of sample
Limited to cells that can be grown in culture
ICAT
Cleavable ICAT
Labeling with acrylamide Proteolysis in the presence of 16 O/18 O Esterification
Derivatization of primary amines MCAT Site-specific photo-cleavable method
Limited to the analysis of proteins containing cysteine Complex MS/MS spectra due to the presence of the affinity label Limited to the analysis of proteins containing cysteine Requires more steps than ICAT Limited to the quantification of proteins containing cysteine Complex analysis. Possible loss or incomplete incorporation of the label Derivatization efficiency might not be high
Applicable to all types of sample Inexpensive, no need for stable isotopes Reduced complexity by enrichment of cysteine-containing peptides Applicable to all types of sample
Requires multiple steps for derivatization increasing the possibility of errors and decreasing the accuracy Derivatization efficiency might not be high Requires multiple steps for derivatization Low accuracy Limited to the analysis of proteins containing cysteine
The most common methods used in quantitative proteomics are reported (left column). None of the above procedures works for all cases and they all have some pros (center column) or cons (right column) that are briefly described.
can be quanti®ed and the information about changes in gene expression are lost. These changes could be quanti®ed only by keeping in consideration the total protein (unmodified modified (e.g. phosphorylated)). On the other hand, an enrichment procedure is necessary for obtaining a better coverage of the phosphoproteome.
Conclusions
Current quantitative proteomics using MS is mainly based on the incorporation of stable isotope tags. Although numerous reports regarding protein quantitation using MS have been published, there are still many areas that require further developments (see Table 1 for a list of pros and cons). In vivo labeling procedures are potentially the most accurate because the label is introduced early in the process; however, it is limited to cells that can be grown in culture. The advantage of the pre-digestion and postdigestion labeling procedures is that they are suitable for all types of sample including human body ¯uids and biopsies. Proteomic technologies are developing at a very fast pace. Some recent efforts, mainly focusing on the identi®cation of proteins from Plasmodium falciparum [49,50], have reported major improvements in throughput. Perhaps the most comprehensive proteome characterization made to date is the identi®cation of 2528 proteins from rice [51]. Current Opinion in Chemical Biology 2003, 7:70±77
In several novel approaches, not described in depth in this review, stable isotopes were not used for the quantitation. For example, Chelius et al. [52] used the intensities in the ion chromatograms from two LC-MS spectra for determining the relative quantities and Stoeckly et al. [53] used the ion intensities from the MALDI spectra of tissue sections for generating semi-quantitative images. Protein chips together with mass spectrometry were also used for expression pro®ling (see [54] for a review). In this case, however, only pro®les without identifying the proteins responsible for the differences were obtained. The differences between samples were characterized as differences in pro®le shapes. Although the idea of obviating the use of stable isotopes is attractive, it seems that the alternatives still need further development before reaching the accuracy usually achieved with stable isotopes.
References and recommended reading
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9.
Kippen AD, Cerini F, Vadas L, Stocklin R, Vu L, Offord RE, Rose K: Development of an isotope dilution assay for precise determination of insulin, C-peptide, and proinsulin levels in non-diabetic and type II diabetic individuals with comparison to immunoassay. J Biol Chem 1997, 272:12513-12522.
10. Wang R, Sweeney D, Gandy SE, Sisodia SS: Detection and quanti®cation of amyloid beta protein and variants by immunoprecipitation-mass spectrometry. J Biol Chem 1996, 271:31894-31902. 11. Oda Y, Huang K, Cross FR, Cowburn D, Chait BT: Accurate quantitation of protein expression and site-speci®c phosphorylation. Proc Natl Acad Sci USA 1999, 96:6591-6596. In this study, the concept of using isotopically labeled media for a quantitative proteomic approach is introduced. The relative abundance of proteins present in the wild type versus mutant yeast cells is reported and the use of this approach for the quanti®cation of phosphorylation sites of the protein Ste20 is described. 12. Smith RD, Pasa-Tolic L, Lipton MS, Jensen PK, Anderson GA, Shen Y, Conrads TP, Udseth HR, Harkewicz R, Belov ME et al.: Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry. Electrophoresis 2001, 22:1652-1658. This study used isotopically enriched media for labeling the proteome of a microorganism and FT-ICR MS for the identi®cation and quanti®cation of the total cell lysate tryptic digest. The same approach was also used for the quanti®cation of intact proteins that were separated by capillary isoelectrofocusing before analysis by FT-ICR. 13. Smith RD, Anderson GA, Lipton MS, Pasa-Tolic L, Shen Y, Conrads TP, Veenstra TD, Udseth HR: An accurate mass tag strategy for quantitative and high-throughput proteome measurements. Proteomics 2002, 2:513-523. Deinococcus radiodurans cells were grown in isotopically enriched media for quantitation. For each identi®ed protein, an accurate mass tag AMT is generated by MS-MS (using an LCQ or FT-ICR instrument) and used for a later high-throughput identi®cation using a single-stage MS. In this study, several proteins were identi®ed and quanti®ed from the tryptic digest of the whole cell lysate. 14. Lipton MS, Pasa-Tolic' L, Anderson GA, Anderson DJ, Auberry DL, Battista JR, Daly MJ, Fredrickson J, Hixson KK, Kostandarithes H et al.: Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags. Proc Natl Acad Sci USA 2002, 99:11049-11054. 15. Conrads TP, Alving K, Veenstra TD, Belov ME, Anderson GA, Anderson DJ, Lipton MS, Pasa-Tolic L, Udseth HR, Chrisler WB et al.: Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine af®nity tags and 15N-metabolic labeling. Anal Chem 2001, 73:2132-2139. www.current-opinion.com
A novel approach that includes growing cells in 15 N-enriched media and labeling cysteines with an af®nity tag for puri®cation of cysteine-containing peptides is described. The cysteine-containing peptides were analysed by LC-FT-ICR MS. They showed that this approach can be used for bacterial cultures as well as mammalian cultures. 16. Washburn MP, Ulaszek R, Deciu C, Schieltz DM, Yates JR III: Analysis of quantitative proteomic data generated via multidimensional protein identi®cation technology. Anal Chem 2002, 74:1650-1657. In this study, a shotgun approach is used for the comparison of two yeast cultures grown in 14 N-minimal media and 15 N-enriched media. These two cultures were mixed and the proteins extracted from the whole cell lysate were digested with trypsin. This complex peptide mixture is ®rst separated by multidimensional LC and then analysed using an LCQ instrument. The identi®cation of more than 800 proteins in a single experiment indicates the great potential of this approach for high-throughput quantitative proteomics. 17. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M: Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 2002, 1:376-386. This study describes an alternative to the procedure of growing cells in 15 N-enriched media. The advantage of this approach is that the use of isotopically labeled leucine is more suitable to cells of mammalian origin. Although it seems to be of general use for culture cells it could not be applied to body ¯uids or tissues. 18. Jiang H, English AM: Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine. J Proteome Res 2002, 1:345. 19. Gu S, Pan S, Bradbury EM, Chen X: Use of deuterium-labeled lysine for ef®cient protein identi®cation and peptide de novo sequencing. Anal Chem 2002, 74:5774-5785. 20. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R: Quantitative analysis of complex protein mixtures using isotope-coded af®nity tags. Nat Biotechnol 1999, 17:994-999. 21. Grif®n TJ, Han DK, Gygi SP, Rist B, Lee H, Aebersold R, Parker KC: Toward a high-throughput approach to quantitative proteomic analysis: expression-dependent protein identi®cation by mass spectrometry. J Am Soc Mass Spectrom 2001, 12:1238-1246. 22. Han DK, Eng J, Zhou H, Aebersold R: Quantitative pro®ling of differentiation-induced microsomal proteins using isotopecoded af®nity tags and mass spectrometry. Nat Biotechnol 2001, 19:946-951. The approach of using ICAT reagents for protein quanti®cation and identi®cation is further extended. In this study, about 500 microsomal proteins are identi®ed and quanti®ed from human myeloid leukemia cells using a combination of ICAT and multidimensional chromatography separation before MS and MS-MS. 23. Grif®n TJ, Gygi SP, Rist B, Aebersold R, Loboda A, Jilkine A, Ens W, Standing KG: Quantitative proteomic analysis using a MALDI quadrupole time-of-¯ight mass spectrometer. Anal Chem 2001, 73:978-986. 24. Smolka M, Zhou H, Aebersold R: Quantitative protein pro®ling using two-dimensional gel electrophoresis, isotope-coded af®nity tag labeling, and mass spectrometry. Mol Cell Proteomics 2002, 1:19-29. This study shows that differential labeling with ICAT reagents can be successfully used in a 2D-PAGE experiment. However the af®nity puri®cation and therefore the af®nity tags are not needed in this kind of experiment and other, simpler procedures would be more suitable. 25. Arnott D, Kishiyama A, Luis EA, Ludlum SG, Marsters JC Jr, Stults JT: Selective detection of membrane proteins without antibodies: a mass spectrometric version of the western blot. Mol Cell Proteomics 2002, 1:148-156. 26. Sechi S, Chait BT: Modi®cation of cysteine residues by alkylation. A tool in peptide mapping and protein identi®cation. Anal Chem 1998, 70:5150-5158. 27. Sechi S: A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis. Rapid Commun Mass Spectrom 2002, 16:1416-1424. Current Opinion in Chemical Biology 2003, 7:70±77
76 Proteomics and genomics
This article further extends the use of deuterated acrylamide to quantitative proteomics. Two complex protein mixtures were separately labeled with acrylamide and deuterated acrylamide before electrophoresis. The ratios between the two isotopic distributions were used for reliably quanti®cation of several proteins present, in ratios from 1:1 to 1:10. 28. Gehanne S, Cecconi D, Carboni L, Righetti PG, Domenici E, Hamdan M: Quantitative analysis of two-dimensional gel-separated proteins using isotopically marked alkylating agents and matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2002, 16:1692-1698. This study con®rms that deuterated acrylamide can be used for quantitation of sample isolated by 2D-PAGE, as previously illustrated. 29. Mirgorodskaya OA, Kozmin YP, Titov MI, Korner R, Sonksen CP, Roepstorff P: Quantitation of peptides and proteins by matrixassisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards. Rapid Commun Mass Spectrom 2000, 14:1226-1232. This study describes the use of 18 O-water for relative quantitation of proteins. Issues relative to the overlap of the two isotopic distributions (one for peptides labeled with 18 O and one for the unlabeled peptides (16 O)), and the possibility of the incorporation of one or two 18 O during the digest, make the calculation of the relative quantities dif®cult. 30. Yao X, Freas A, Ramirez J, Demirev PA, Fenselau C: Proteolytic 18 O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Anal Chem 2001, 73:2836-2842. The use of 18 O-water previously described by Mirgorodskaya et al. [29] is further extended for the comparison of two adenovirus serotypes. Although the use of a high-resolution instrument like an FT-ICR solves the problems related to the overlap of the two isotopic distributions, it seems that the calculation of the relative quantities is still complex. 31. Goodlett DR, Keller A, Watts JD, Newitt R, Yi EC, Purvine S, Eng JK, von Haller P, Aebersold R, Kolker E: Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation. Rapid Commun Mass Spectrom 2001, 15:1214-1221. The relative quantities of proteins are determined by differential labeling of the tryptic digest of two samples with methanol and D3-methanol. This procedure of permethylation has the added bene®t of facilitating de novo sequencing. 32. Ji J, Chakraborty A, Geng M, Zhang X, Amini A, Bina M, Regnier F: Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J Chromatogr B Biomed Sci Appl 2000, 745:197-210. 33. Munchbach M, Quadroni M, Miotto G, James P: Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety. Anal Chem 2000, 72:4047-4057. Complex protein mixtures were separated by 2D- or 1D-PAGE and the spots to be identi®ed/quanti®ed were selected from two gels (e.g. treated and control). The proteins were succinilated with a heavy (D4) or light (D0) reagent before digestion with Asp/Glu-C. The conditions for succinilation of the lysine were improved and optimized to avoid secondary unwanted reactions. An added bene®t of this procedure is that the labeling of the Nterminus facilitates de novo sequencing because the b-ion intensities are greatly increased. Although this approach has several advantages, it does not overcome the issue of reproducibility that is inherent to 2DE. 34. Zhang R, Sioma CS, Wang S, Regnier FE: Fractionation of isotopically labeled peptides in quantitative proteomics. Anal Chem 2001, 73:5142-5149. In this study, the elution times between the heavy and the light forms of acetylated peptides are compared with the retention time of peptides modi®ed with ICAT reagents. Acetylated peptides co-elute independently from the label, whereas the light and heavy forms of the ICAT-modi®ed peptides have different retention times. 35. Wang S, Zhang X, Regnier FE: Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates. J Chromatogr A 2002, 949:153-162. The novelty of this procedure is that a puri®cation step for isolating cysteine- and/or histidine-containing peptides is used. The quanti®cation procedure is the same as previously described for succinic anhydride. The approach seems reasonable, although the copper af®nity column might not be exclusively selecting for histidine-containing peptides. 36. Liu P, Regnier FE: An isotope coding strategy for proteomics involving both amine and carboxyl group labeling. J Proteome Res 2002, 1:443. Current Opinion in Chemical Biology 2003, 7:70±77
37. Kindy JM, Taraszka JA, Regnier FE, Clemmer DE: Quantifying peptides in isotopically labeled protease digests by ion mobility/ time-of-¯ight mass spectrometry. Anal Chem 2002, 74:950-958. 38. Cagney G, Emili A: De novo peptide sequencing and quantitative pro®ling of complex protein mixtures using mass-coded abundance tagging. Nat Biotechnol 2002, 20:163-170. MCAT is introduced. Lysine guanidination, previously employed to facilitate sequencing by post-source decay (PSD), was used to facilitate sequencing by LC-MS/MS. The comparison of the isotopic distribution of unlabeled peptides with guanidinilated peptides was used for relative quantitation of yeast proteins. Although the average errors are within 25%, some peptides have errors as big as 62%, perhaps due to changes in ionization between lysine and guanidil-lysine. 39. Zhou H, Ranish JA, Watts JD, Aebersold R: Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat Biotechnol 2002, 20:512-515. This study describes a procedure for simultaneous solid-phase enrichment and labeling of cysteinyl peptides. The method described seems to be a signi®cant improvement to the typical ICAT approach. 40. Ruse CI, Willard B, Jin JP, Haas T, Kinter M, Bond M: Quantitative dynamics of site-speci®c protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry. Anal Chem 2002, 74:1658-1664. 41. Stemmann O, Zou H, Gerber SA, Gygi SP, Kirschner MW: Dual inhibition of sister chromatid separation at metaphase. Cell 2001, 107:715-726. In this article, labeled synthetic peptides were used as internal standards in an LC-MS experiment for quantifying the grade of phosphorylation of separase, a protein involved in chromatid separation during the anaphase. 42. Oda Y, Nagasu T, Chait BT: Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotechnol 2001, 19:379-382. 43. Adamczyk M, Gebler JC, Wu J: Selective analysis of phosphopeptides within a protein mixture by chemical modi®cation, reversible biotinylation and mass spectrometry. Rapid Commun Mass Spectro 2001, 15:1481-1488. 44. Zhou H, Watts JD, Aebersold R: A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 2001, 19:375-378. 45. Goshe MB, Veenstra TD, Panisko EA, Conrads TP, Angell NH, Smith RD: Phosphoprotein isotope-coded af®nity tags: application to the enrichment and identi®cation of lowabundance phosphoproteins. Anal Chem 2002, 74:607-616. Phospho Ser/Thr were labeled with EDT-D0 or EDT-D4 after b-elimination. The proof of principle is done using casein. In theory, this approach could be used for the quantitation of phosphorylation sites on a proteomic scale. However, using the described method, O-glycosylation sites would be also labeled and the procedure for avoiding the labeling of these sites has not been developed. 46. Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM: Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 2002, 20:301-305. This study describes a methodology for phosphoproteome characterization. Although it has not been used for quantitation purposes, this enrichment procedure is of general use for phosphopeptides. More than 1000 phosphopeptides were detected and 383 phosphorylation sites identi®ed in one experiment from yeast cell lysate. 47. Pandey A, Podtelejnikov AV, Blagoev B, Bustelo XR, Mann M, Lodish HF: Analysis of receptor signaling pathways by mass spectrometry: identi®cation of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc Natl Acad Sci USA 2000, 97:179-184. 48. Pandey A, Fernandez MM, Steen H, Blagoev B, Nielsen MM, Roche S, Mann M, Lodish HF: Identi®cation of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways. J Biol Chem 2000, 275:38633-38639. 49. Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL et al.: A proteomic view of the Plasmodium falciparum life cycle. Nature 2002, 419:520-526. www.current-opinion.com
Quantitative proteomics using mass spectrometry Sechi and Oda 77
50. Lasonder E, Ishihama Y, Andersen JS, Vermunt AM, Pain A, Sauerwein RW, Eling WM, Hall N, Waters AP, Stunnenberg HG, Mann M: Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 2002, 419:537-542. 51. Koller A, Washburn MP, Lange BM, Andon NL, Deciu C, Haynes PA, Hays L, Schieltz D, Ulaszek R, Wei J et al.: Proteomic survey of metabolic pathways in rice. Proc Natl Acad Sci USA 2002, 99:11969-11974. 52. Chelius D, Bondarenko PV: Quantitative pro®ling of proteins in complex mixtures using liquid chromatography and mass spectrometry. J Proteome Res 2002, 1:317. Determination of quantities base only on ion intensities from LC-MS spectra. This seems a preliminary but promising approach.
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53. Stoeckli M, Chaurand P, Hallahan DE, Caprioli RM: Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nat Med 2001, 7:493. This article elegantly describes the use of MS for imaging. The image is generated by MALDI MS, scanning a tissue section imbedded with matrix. Several mouse brain sections are used to illustrate the technology. 54. Weinberger SR, Dalmasso EA, Fung ET: Current achievements using ProteinChip Array technology. Curr Opin Chem Biol 2002, 6:86-91. This article reviews the use of ProteinChip array for quantitation using surface enhanced laser-desorption (SELDI) protein chips.
Current Opinion in Chemical Biology 2003, 7:70±77