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Molecular & Cellular Proteomics 1:19-29, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Research
Quantitative Protein Profiling Using Two-dimensional Gel Electrophoresis, Isotope-coded Affinity Tag Labeling, and Mass Spectrometry*
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Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Sao Paulo 13083-970, Brazil
Institute for Systems Biology, Seattle, Washington 98105
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.
Proteomics attempts to study the structure, function, and control of biological systems and processes by the systematic and quantitative analysis of the many properties of proteins. These include the sequence (identity), abundance, activity, and structure of the proteins expressed in a cell, as well as the modifications, interactions, and translocations each protein might experience. There is currently no single experiment or platform that permits the analysis of all these properties on a proteome-wide scale. Therefore, technologies and platforms have been developed that specifically study a subset of these properties.
The systematic identification of the proteins contained in a complex sample and the determination of the relative abundance of each protein (quantitative protein profiling) if two or more samples are being compared are central objectives of proteomics. For this purpose two methods have been developed and are routinely being used. The first is the combination of the well established techniques of two-dimensional gel electrophoresis (2DE)1 and mass spectrometry (MS) (reviewed in Ref. 1). The second is a recently developed procedure based on isotope-coded affinity tag (ICATTM) reagent protein labeling, liquid chromatography, and tandem mass spectrometry (2, 3).
At present the 2DE/MS method is most commonly used. Proteins separated by 2DE and detected by staining are identified, one-by-one, by mass spectrometric analysis of peptide fragments derived from each protein. The method is supported by robust and automated instruments that perform specific steps in the process, such as gel imaging, spot picking, protein digestion, peptide mass spectrometry, and sequence data base searching (1, 4–6). In this method protein quantification is achieved by image analysis of the spot patterns generated by staining the proteins separated in the gel, implicitly assuming that the staining intensity of a protein spot accurately indicates the amount of protein contained in the spot. Unfortunately, the practical implementation of this simple concept has been difficult, mainly because of limitations of the available staining and gel electrophoresis methods. These include limited dynamic range, sensitivity, and reproducibility, respectively, of the popular Coomassie Blue and silver staining procedures (7, 8), limited pattern reproducibility of 2DE gels, and a recurrence of several proteins to migrate to the same gel coordinates. The development of sensitive fluorescence staining methods with a linear signal response over a wide dynamic range (9, 10) and the introduction of a two-color fluorescent labeling system allowing the concurrent electrophoresis of two differentially labeled protein samples in the same 2DE gel and thus the determination of the ratio of abundance by spectral analysis of each protein spot (11, 12) have eliminated or at least alleviated some of the challenges related to 2DE gel imaging.
As 2DE gel-based proteomic studies depend on the analysis of the separated proteins by MS, it has been suggested to use stable-isotope dilution to achieve accurate protein quantification in the mass spectrometer, obviating the need for gel imaging (13, 20). In these experiments proteins were metabolically labeled with stable isotopes (15N or 13C), and two samples with distinguishable isotope signatures were combined and concurrently separated by 2DE. Proteins in specific spots were then digested, and the resulting peptides were extracted and analyzed by MS. The ratio of signal intensities of the isotopically normal and heavy forms of a particular peptide was then used to calculate the ratio of abundance of the respective protein. Although quantification was shown to be very accurate, this method is limited to cells that can be cultured in isotopically defined culture conditions.
In this paper we show that post-isolation isotopic protein labeling using the ICATTM reagents is also compatible with 2DE and accurate protein quantification by MS. We show that proteins labeled with the isotopically heavy and normal forms of the reagent, respectively, precisely co-migrate in 2DE gels and that the ratio of protein abundance can be accurately determined from the relative mass spectrometric signal intensities of the heavy and normal forms of labeled, cysteine-containing peptides. Both identification and accurate quantification are therefore achieved in a simple single-step analysis. This method also reliably identifies and quantifies each protein in spots containing multiple different polypeptides and identifies and quantifies each isoform or post-translationally modified form of a protein that migrates to different positions. As the method is based on post-isolation chemical labeling of proteins, it is compatible with most if not all protein samples that can subsequently be separated and analyzed by 2DE.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Labeling Standard Proteins—
The standard proteins used were bovine serum albumin (BSA), chicken ovalbumin (OVAL), bovine ß-lactoglobulin (LACB), bovine
-lactalbumin (LCA), or superoxide dismutase (Sigma). Labeling was performed as described previously (14), with slight modifications. 1 mg of each protein was dissolved in 1 ml of Solution A (0.05% (w/v) SDS, 6 M urea, 5 mM EDTA, and 50 mM Tris buffer, pH 8.3). Specified amounts of each protein solution were mixed together (as described for each case under "Results"), and aliquots of 100 µg of total protein mixture (100 µl) were labeled. Disulfide bonds were reduced with 5 mM tributyl phosphine (Sigma) for 10 min and then 60 µg of the isotopically normal or heavy form of ICATTM reagent (Applied Biosystems, Framingham, MA) was added. This amount of ICATTM reagent (105 nmol) represented an 
1 mM concentration and a 3 to 1 molar ratio over total protein sulfhydryls assuming an average molecular protein mass of 30 kDa and 8 cysteine residues per protein. The reaction mixtures were incubated for 2 h at room temperature in an Eppendorf tube in the dark. The reactions were stopped by adding DTT to a final concentration of 10 mM. The light and heavy ICATTM reagent-labeled protein mixtures were combined prior to 2DE separation. Incomplete labeling of proteins was carried out under the conditions described above except that the sample was diluted 10-fold, and urea was omitted.
Preparation and Labeling Yeast Protein Extracts—
Yeast cells (Saccharomyces cerevisiae strain BWG1-7A) were grown until mid-log phase in yeast/peptone/dextrose medium containing either 2% (w/v) glucose or 2% (w/v) galactose as carbon source and harvested by centrifugation. Glucose- and galactose-grown cells were lysed separately in lysis buffer containing 1% (w/v) SDS, 50 mM Tris, pH 8.0, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride after lyticase treatment to prepare spheroblasts according to standard procedures (15). An aliquot of each of the resulting protein extracts was used to determine the protein concentration using a commercial protein assay kit (Bio-Rad). To the remainder of the samples DTT was added to a final concentration of 100 mM, and the solution was boiled for 5 min. 300 µg of total protein from each extract was precipitated with a cold ethanol:acetone:acetic acid (50:50:0.1 (v/v/v)) solution, and the precipitate was washed once with 70% ethanol in water. The protein pellet was re-suspended in 50 µl of Solution A, and the disulfide bonds were reduced by the addition of tributyl phosphine to a final concentration of 5 mM and incubation for 10 min at room temperature. 100 µg of ICATTM reagent (3 mM final concentration) was added to the reaction solution. After 2 h the reaction was stopped by adding DTT to a final concentration of 10 mM.
For the purpose of comparing 2DE gel patterns of each sample (glucose- or galactose-grown) the labeled proteins were again precipitated as described above, re-dissolved, and separated by 2DE. For concurrent electrophoresis of both samples on the same gel and subsequent quantification by MS, equal amounts of the two labeled extracts were combined, precipitated as described above, re-dissolved, and separated by 2DE.
Two-dimensional Gel Electrophoresis—
Approximately 20 µg of standard protein mixture or 300 µg of yeast protein extract were solubilized in 340 µl of sample buffer containing 8 M urea, 4% (w/v) CHAPS, 2% (v/v) carrier ampholytes, pH 3–10, 70 mM DTT, and 0.001% (w/v) bromphenol blue. Except for CHAPS, which was from Sigma, all reagents were from Amersham Biosciences, Inc. Samples were applied to immobilized pH gradient strips with a non-linear separation range of pH 3–10 (catalog number 17-1235-01; Amersham Biosciences, Inc.). After a 10-h rehydration, isoelectric focusing was carried out, at 20 °C, for 1 h at 500 V, for an additional hour at 1000 V, and then for 10 h at 8000 V in an IPGphor apparatus (Amersham Biosciences, Inc.) maintaining a limiting current of 50 µA per strip. First dimension strips were subjected to the standard reduction and alkylation steps prior to second-dimension electrophoresis. Strips were soaked for 10 min in a solution containing 50 mM Tris-HCl, pH 6.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 2% (w/v) DTT and for an additional 10 min in the same solvent containing 2.5% (w/v) iodoacetamide instead of DTT. Second-dimension electrophoresis (SDS-PAGE) was performed on an SE 600 system connected to a MultiTemp II refrigerating system (both from Amersham Biosciences, Inc.). After laying the strip on the top of a 12.5% polyacrylamide gel and sealing it with agarose, electrophoresis was carried out for 1 h at 90 V, at which time a constant amperage of 30 mA per gel was applied until the dye front reached the lower end of the gel. Proteins were detected by a silver nitrate staining protocol adapted from Ref. 16.
Protein Identification and Quantitation by Matrix-assisted Laser Desorption Ionization (MALDI) Quadrupole Time-of-Flight (TOF) MS—
Peptides were generated and extracted from the gel-separated proteins following established in-gel tryptic digestion protocols (17). The peptides were analyzed on a QSTARTM Pulsar quadrupole TOF tandem mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada), which was equipped with a MALDI ion source. Prior to application to the sample plate the samples were purified using C18 ZipTips (Millipore) and mixed at a 1:1 ratio with a solution of 16% (w/v) 2,5-dihydrobenzoic acid in acetonitrile:water (25:75 v/v).
Identification of the standard proteins was performed by submitting its known sequences to the Peptidemass tool (18) and manually comparing the predicted peptide masses of each standard protein to the measured masses in the mass spectrum. For identification of the proteins from yeast extracts, the measured mass of the tryptic peptides were searched against S. cerevisiae entries from Swiss-Prot using the MS-Fit program (University of California, San Francisco; prospector.ucsf.edu/) (19). In this program, it is already possible to consider ICATTM labeling of cysteines for calculating the predicted mass of the peptides for each protein. The observed pI and MW of the identified spots in the 2DE map were compared with the theoretical pI and MW to confirm identifications.
For quantifying the relative abundance of a protein, the peak height of the monoisotopic peak of the light ICATTM-labeled peptide was divided by the peak height of the monoisotopic peak of the heavy labeled form of the peptide. When more than one cysteine-containing peptide was detected for a particular protein, the abundance ratio was calculated from the peak pair with the highest signal intensity. In cases in which the shape of the isotope distributions of the light and heavy ICATTM–labeled peptides differed (i.e. asymmetric shape of the clusters) we suspected the presence of contaminating peaks and did not use such peak pairs for quantification. Instead, the peak pair with the next highest signal intensity was used.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Principle of the Method—
The method for quantitative protein profiling that we describe in this paper is schematically illustrated in Fig. 1. It is based on the separation of proteins labeled with ICATTM reagents by 2DE and their identification and quantification by mass spectrometry. The proteins contained in two separate samples are first labeled with the isotopically normal (non-deuterated) or heavy forms (deuterated), respectively, of ICATTM reagents according to established protocols (2, 14).2 The two samples are then combined and concurrently separated by 2DE in the same gel, and the separated proteins are detected by an MS compatible staining protocol. The proteins migrating to specific spots are enzymatically digested in the gel matrix, and the resulting peptides are extracted and analyzed by mass spectrometry. Protein identification is achieved either by peptide mass mapping in a single-stage mass spectrometer (21, 22) or by collision-induced dissociation of selected peptides and sequence data base searching (23). The ratio of abundance of the protein(s) in the spot analyzed is determined by the ratio of signal intensities for the isotopically normal and heavy forms of a specific, tagged peptide.
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Migration of ICATTM Reagent-labeled Proteins in 2DE—
To determine whether and how labeling with the ICATTM reagent affected protein migration during 2DE a mixture of equal amounts of LACB and LCA was labeled with the isotopically normal form of ICATTM reagent and separated by 2DE. The 2DE profile of the labeled proteins was compared with the profile obtained from an unlabeled but otherwise identical sample separated under the same conditions. Fig. 2, A and B show the profiles obtained from the unlabeled and labeled samples, respectively. It is apparent that ICATTM reagent labeling decreased the electrophoretic mobility of both proteins. The observed mobility decrease was consistent with the mass of 442 Da added by each ICATTM reagent molecule. For LCA, which contains eight cysteines, the calculated molecular mass increased from 14186 to 17722 Da, and the apparent molecular mass increased from 14 to
18 kDa. For LACB, which contains five cysteines, the calculated molecular mass increased from 18281 to 20491 Da, and the apparent molecular mass increased from 18 to 21 kDa. In contrast, no shift in pI was apparent as a consequence of ICATTM labeling. The observation that the pI of the acidic proteins LCA and LACB is unaffected is consistent with the fact that the ICATTM reagent molecule is uncharged and, by reacting with sulfhydryl groups that are uncharged at acidic pH values, does not alter the solution charge state of the protein. For basic proteins a slight change in pI is expected to occur after ICATTM reagent labeling, because the negative charge of sulfhydryl groups at pH values above the pK of sulfhydryls will be neutralized by the labeling reaction.
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We have demonstrated previously that incomplete labeling of proteins with ICATTM reagents results in a "fuzzy" banding pattern in SDS-PAGE and reported optimized labeling protocols for complete labeling of all the cysteines in a reduced protein (14). Fig. 2C shows the 2DE profile of partially labeled LCA and LACB if a non-optimized labeling protocol was used. It is evident that incomplete protein labeling is easily recognized by the characteristic ladder spot pattern and that the well defined spots achieved by complete labeling are essential for the success of the method.
Co-migration by 2DE of Proteins Labeled with Isotopically Normal and Heavy ICATTM Reagents—
To determine whether proteins labeled with the different isotopic forms of the ICATTM reagent migrated to identical coordinates in 2DE gels, samples of LCA were labeled with the isotopically normal or heavy form of the reagent. The proteins were combined at a d0:d8 (light ICATTM: heavy ICATTM) ratio of 11:1, and the mixed sample was separated by 2DE. The resulting spot, detected by silver staining, was divided into four quadrants, and the protein in each quadrant was in-gel digested and analyzed mass spectrometrically. Fig. 3A shows the gel spot, and the four quadrants and Fig. 3B detail the d0:d8 ratios used for quantification. The expected d0:d8 ratio was 11.00. The ratio calculated by averaging the observed values over all the quadrants was 11.72. The highest deviation from this observed mean value detected in quadrant number 3 was 11.30, corresponding to a 3.6% difference.
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We chose LCA for this experiment, because the prevalence of cysteine residues in this protein would be expected to exaggerate isotope effects on electrophoretic migration. At eight cysteine residues the heavy ICATTM reagent-labeled protein contained 64 deuterium atoms. Moreover, the pI (4.8) and molecular mass (14.1 kDa) of LCA position this protein in an area of high resolution in the gel used. These results indicate that proteins labeled with differentially isotopically labeled forms of the ICATTM reagents precisely co-migrate by 2DE.
Accuracy of Quantification—
To evaluate the accuracy of quantification of the method we prepared and analyzed two protein mixtures containing the same five proteins at different quantities. The composition of the samples is indicated in Table I. One sample was labeled with the isotopically normal and the other with the heavy form of the ICATTM reagent, and the samples were combined and separated by 2DE. Protein spots were detected by silver staining and in-gel digested with trypsin, and the resulting peptides were identified and quantified by mass spectrometry.
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The concise shape of the protein spots in the 2D electropherogram and the absence of vertical "streaking" suggested that quantitative labeling had been achieved (Fig. 4A). The multiple spots (with same MW and different pI) observed for LACB, OVAL, and BSA were also observed in the 2DE map of the unlabeled protein mixture (data not shown) and are therefore unrelated to the labeling with ICATTM reagent. The identity of each protein spot was confirmed by peptide mass fingerprinting. For each protein, the predicted masses of the labeled, cysteine-containing peptides were also calculated and used to identify the corresponding peaks in the mass spectra. Fig. 4B shows the peptide mass spectrum of superoxide dismutase and expansions of the peak areas containing the signals for cysteine-containing peptides. Each peptide appeared as two isotope envelopes with an 8.04-Da mass difference, corresponding to the non-deuterated and deuterated forms of the peptides. Fig. 4C shows the peaks of cysteine-containing peptides used for quantification of the other four proteins in the mixture. The signal intensities of the monoisotopic peaks were used to calculate the observed abundance ratios. The measured ratios are indicated and related to the expected respective ratios in Table I. The discrepancy between the expected and measured values was less than 12% for all the proteins in the mixture. These results demonstrate the possibility of using ICATTM reagent labeling for accurate quantification of proteins separated by 2DE.
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Quantification of Changes in Protein Abundance in Yeast Induced by Metabolic Shift—
Protein expression in yeast is known to be highly affected by the type of carbon source used. To assess the ability of the method of this paper to identify proteins in complex mixtures and to quantify changes in their abundance, we investigated changes in the protein profile of S. cerevisiae induced by a shift from glucose to galactose in the carbon source. A protein extract from cells grown in glucose-containing medium was labeled with the isotopically normal ICATTM reagent, and an extract from yeast grown in galactose-containing medium was labeled with the heavy ICATTM reagent. The samples were either run individually (see Fig. 5, A for glucose and B for galactose) or combined (Fig. 6) in 2DE gels. No indication for partial protein labeling was detectable. Moreover, the quality of the 2DE gels was comparable with that of 2DE gels of unlabeled yeast extracts (data not shown), indicating that ICATTM labeling of proteins does not interfere with the separation of complex protein mixtures.
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Thirteen spots from the 2DE map presented in Fig. 6 were in-gel digested and submitted to identification and quantification by mass spectrometry. The results are summarized in Table II. The abundance ratios indicated were calculated from the intensities of the most intense ICATTM reagent-labeled peptides. The expression level of metabolic enzymes such as adolase, phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase 3 was observed to be lower in the galactose-grown yeast, a result consistent with results obtained from microarray experiments (24). In contrast, mitochondrial proteins such as COX4 and ATPD were found to be de-repressed in the absence of glucose, also consistent with the mRNA data.
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Quantitative Analysis of Post-translational Modifications—
Post-translational modifications frequently change protein pI and/or apparent MW of proteins and are therefore easily detected by 2DE. To assess the potential of the method to detect quantitative changes in the spot pattern of differentially modified proteins and therefore to quantify induced changes in protein modification we mass spectrometrically analyzed individual spots in the spot pattern of ENO2 and HSP70, proteins known to be phosphorylated (25, 26). Results are shown in Fig. 7. The three ENO2 spots showed a consistent glucose:galactose ratio of 2.4:1. In contrast, the ratio for the two HSP70 spots analyzed varied significantly (2.7:1 for the more acidic spot; 1:1 for the more basic spot) indicating that the modification causing the acidic shift is increased if the cells are grown in glucose. Although the observed spot pattern was consistent with protein phosphorylation, no phosphorylated peptide was detected directly by MS. For the positive identification of phosphorylated peptides auxiliary methods such as metabolic [32P] or [33P] radiolabeling or immunodetection (27, 28) might need to be employed. These results indicate that the method can detect quantitative changes in protein modification or processing, provided that the different protein forms can be separated by 2DE.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with ICATTM reagents by 2DE and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different ICATTM reagents precisely co-migrate during 2DE and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. The ICATTM–labeled proteins can be separated by 2DE at a resolution comparable with unlabeled protein samples. We show that the method has a quantitation accuracy of better than 20% and that different isoforms or the differentially post-translationally modified forms of a protein can be identified individually and quantified. Furthermore, different proteins migrating to the same 2DE coordinates can also be identified and quantified individually.
For separating ICATTM reagent-labeled proteins at high resolution it was critical that the labeling reaction proceeded to completion. Incomplete labeling was evident from vertically streaking spots. Based on the absence of detectable vertical streaks, the labeling procedures we and others have optimized previously (14)2 achieved near quantitative labeling even for very complex samples. ICATTM reagent concentrations in excess of 1 mM (which results in a molar excess of reagent over to the sulfhydryl groups), the addition of urea, and the maintenance of protein solubility by the addition of SDS were shown to significantly improve the labeling reaction.
Langen et al. (20) and Oda et al. (13) have used metabolic stable isotope labeling of proteins prior to 2DE, also for the purpose of quantifying the proteins by mass spectrometry. Metabolic stable isotope labeling, although simple and effective, is only applicable to protein samples from cells that can be cultured in isotopically enriched or depleted media and is therefore essentially restricted to microbial species. Furthermore, the number of heavy isotopes added by metabolic labeling is sequence-specific. The mass difference between pairs of signals representing the heavy and normal form of a specific peptide is therefore variable, complicating protein identification and quantification. In contrast, the ICATTM reagent labeling method is based on post-isolation chemical tagging of proteins and is therefore compatible with essentially any protein sample that contains cysteine. Furthermore, with the exception of the cysteine-containing peptides, peptide masses remain unchanged. Protein identification by peptide mass fingerprinting, the detection of post-translationally or otherwise modified peptides in the sample, and the detection of the peptides tagged with the ICATTM labeling reagent is therefore a straightforward operation. Because cysteine is a relatively rare amino acid, the positive identification of cysteine-tagged peptides in a peptide mixture provides a strong constraint for sequence data base searching (29). Cysteine-containing peptides are easily identified by the presence of the different isotopic forms. Data base search programs such as MS-fit (19) already include ICATTM reagent labeling as a possible cysteine modification. The use of post-isolation isotopic protein labeling prior to 2DE/MS analysis therefore presents a simpler and more general alternative to metabolic stable isotope labeling.
Recently, an elegant method was introduced (11) and evaluated (12) in which proteins are covalently labeled with fluorescent dyes prior to separation by 2DE. Two structurally similar dyes with different spectral properties were used to label the proteins in two samples with a specific color, the combined samples were concurrently separated by 2DE, and the ratio of abundance for spot was determined by spectral analysis of the emitted fluorescent light. This method also effectively eliminates the problem of electrophoretic variation between gels, experiments demonstrating accurate quantification have validated the technique (11, 12), and fluorescent detection is potentially extremely sensitive. A drawback of the current implementation is that the epsilon amino groups of the abundant amino acid lysine are labeled with relatively hydrophobic dyes, thus reducing the solubility of extensively labeled proteins. To avoid protein precipitation in the gel, minimal labeling is performed. This incomplete labeling procedure limits the sensitivity of the method and makes equal quantitative labeling between two samples more difficult. Another problem is that the labeled and unlabeled proteins of the same species migrate to different coordinates in the 2DE gel, generating a rather complex 2DE profile.
In contrast, the method described in this paper is based on complete protein labeling. Consequently, all of the protein molecules of a particular species are concentrated in the same spot. Furthermore, the present method offers the possibility of individually quantifying multiple proteins migrating to the same spot, a feature that is impossible with the fluorescent or other imaging techniques. We believe that the present method should represent an attractive alternative to the fluorescent labeling method, particularly in cases in which mass spectrometric analysis of the separated proteins is attempted.
In addition to being a constraining factor in sequence data base searching cysteine-specific protein labeling has other beneficial features. As cysteine is relatively rare no extreme shifts in SDS-PAGE migration are expected after labeling. We also did not notice a decrease in protein solubility after ICATTM reagent labeling, probably because the amino acids with charged side chains (lysine, arginine, glutamic acid, and aspartic acid) are not affected by the labeling reaction and because the reagent used is not very hydrophobic. It was also of practical importance that all the cysteines were reacted with ICATTM reagent molecules, thus eliminating the problem of cysteine re-oxidation during the IEF run and contributing to maintain the proteins in a desirable denatured state. In most 2DE protocols, cysteine reduction is achieved by including high DTT concentrations to the IEF separation solution. But during the IEF run, DTT may acquire a negative charge and migrate to the anode, depleting reducing power on the basic regions. Moreover, DTT accumulated in the acidic region may migrate in the second-dimension run and cause artifacts in the 2DE profile. Labeling proteins with ICATTM prior to IEF separation should avoid the necessity of using DTT in the IEF run. The subsequent reduction/alkylation step prior to the second-dimension run can also be eliminated.
The described technique has several limitations. The ICATTM reagent method using current reagents only labels cysteine-containing proteins. For the S. cerevisiae proteome 92% of the proteins contain at least one cysteine residue. Although any other protein would also be identified by the method, accurate quantification relies on the detection of at least one cysteine-containing peptide in the mass spectrum. Reagents with different specificities that are under development will eliminate this limitation. In the described method protein quantification is achieved in the mass spectrometer on individual spots. Therefore, for determining differentially expressed spots it is either necessary to run three gels per sample, one for each of the samples separately and one for the combined sample, or to analyze all the spots present in the gel containing the combined samples. With the increasing availability of automated, high throughput robotic sample-processing systems (4–6) and the development of methods for parallel isolation and digestion of 2DE-separated proteins (30, 31) the latter option is increasingly becoming feasible.
Concluding Remarks—
Significant advances in protein identification have been accomplished over the last ten years through the use of mass spectrometry, and large-scale or even proteome-wide protein identification is now a common practice. The descriptive information obtained from such protein cataloguing projects can be significantly enhanced if the quantity and changes thereof can be determined precisely for each protein in a sample. The strategy described in this paper provides a simple and effective tool by which accurate protein quantitation can be achieved in proteome-wide experiments. The method also allows the determination of the absolute 2DE and mass spectrometry, two techniques established in most proteomics laboratories. The method also lends itself to determine the absolute amounts of specific proteins, if isotopically labeled, calibrated internal standards are being used and for the selective, quantitative analysis of selected protein spots, a task that might be very useful for medical diagnostic purposes. We therefore anticipate that this method will find wide application in the field of quantitative proteomics.
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ACKNOWLEDGMENTS |
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The support and critical input from Drs. Ken Parker and George Vella (Applied Biosystems, Framingham, MA) are gratefully acknowledged.
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FOOTNOTES |
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Received, September 5, 2001, and in revised form, October 9, 2001.
Published, October 11, 2001
1 The abbreviations used are: 2DE, two-dimensional (isoelectric focusing/SDS-PAGE) gel electrophoresis; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; ICATTM, isotope-coded affinity tag reagents; IEF, isoelectric focusing; LACB, bovine ß-lactoglobulin; LCA, bovine -lactalbumin; MALDI, matrix-assisted laser desorption-ionization; MS, mass spectrometry; MW, molecular weight; OVAL, chicken ovalbumin; TOF, time-of-flight. 
2 T. Nadler, K. Parker, B. Wagenfeld, R. Lotti, B. Purkayastha, S. Daniels, W. Stanick, S. Pillai, J. N. Marchese, and G. Vella (2001) Optimization of a protocol for preparing protein samples with an isotope-coded affinity tag (ICATTM) reagent, submitted for publication.
* This work was supported in part by NCI, National Institutes of Health Grant 1R33CA84698 (to R. A.) and by a fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (to M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Inst. for Systems Biology, 4225 Roosevelt Way, Suite 200, Seattle, WA 98105. Tel.: 206-732-1204; Fax: 206-732-1254; E-mail: raebersold{at}systemsbiology.org.
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