Anal. Chem. 2001, 73, 4891-4902
Peptide Mass Mapping Constrained with Stable Isotope-Tagged Peptides for Identification of Protein Mixtures Thomas C. Hunter,† Li Yang,† Haining Zhu,† Vahid Majidi,† E. Morton Bradbury,†,‡ and Xian Chen*,†
C-ACS, BN-2, MS M888, Chemistry Division, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87544, and Department of Biological Chemistry, School of Medicine, University of California at Davis, Davis, California 95616
Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PMM) can be generated for protein identification. However, insufficient peptide mass accuracy and protein sequence coverage limit the potential of the PMM approach for highthroughput, large-scale analysis of proteins. In our novel approach, nonlabile protons in particular amino acid residues were replaced with deuteriums to mass-tag proteins of the S. cerevisiae proteome in a sequencespecific manner. The resulting mass-tagged proteolytic peptides with characteristic mass-split patterns can be identified in the data search using constraints of both amino acid composition and mass-to-charge ratio. More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identify the parent proteins at modest mass accuracy and low sequence coverage. As a result, the specificity and accuracy of a PMM was greatly enhanced without the need for peptide sequencing or instrumental improvements to obtain increased mass accuracy. The power of PMM has been extended to the unambiguous identification of multiple proteins in a 1D SDS gel band including the identification of a membrane protein. Mass spectrometry (MS) has recently become an essential tool for protein analysis on a genomic scale, i.e., proteomics, because of its speed, sensitivity, and accuracy.1-6 There are two popular MS-based proteomic approaches: (i) two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) protein separation, proteolysis, and determination of the peptide masses by matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) MS3,7 or electrospray ionization (ESI) MS; (ii) liquid chromatog* To whom correspondence should be addressed. Tel: 505-665-0781. Fax: 505-665-3024. E-mail:
[email protected] . † Los Alamos National Laboratory. ‡ University of California at Davis. (1) Blackstock, W. P.; Weir, M. P. Trends Biotechnol. 1999, 17, 121-127. (2) Yates, J. R., III. J. Mass Spectrom. 1998, 33, 1-19. (3) Shevchenko, A.; Jensen, O. N.; Podtelejnikov, A. V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Boucherie, H.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445. (4) Smith, R. Nat. Biotechnol. 2000, 18, 1041-1042. (5) Keogh, T.; Lacey, M. P.; Fieno, A. M.; Grant, R. A.; Sun, Y.; Bauer, M. D.; Begley, K. B. Electrophoresis 2000, 21, 2252-2265. (6) Siuzdak, G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11290-1129. 10.1021/ac0103322 CCC: $20.00 Published on Web 09/15/2001
© 2001 American Chemical Society
raphy (LC) separation coupled with ESI MS.5,8-10 These approaches measure the mass-to-charge ratios (m/z) of proteolytic peptides to provide the peptide mass map (PMM) or fingerprint of proteins. Unique proteins can be identified by searching for the best matches between the experimental and theoretical PMMs derived from the translated genomic sequence or expressed sequence tags (EST).11-18 Whereas PAGE gels are most commonly used to separate the proteins from cellular extracts, the recovery of proteolytic peptides from the in-gel digestion of protein spots suffers from low efficiencies of both protein digestion and peptide extraction, leading to low sequence coverage, i.e., a low number of detectable peptides from a protein.7 Further, the variability in peptide ionization efficiency also contributes to the problem.19 Usually, the proteolytic sequence coverage of a protein is much less than 30%, which is insufficient for the unambiguous identification of a protein directly from a PMM.16 Additional spectral complications such as mass degeneracy, chemical modification, and ion suppression become more pronounced with increasing numbers of proteolytic peptides from multiple components or large protein sizes in a given spot.15,18 When the m/z of peptides is the sole parameter in a PMM, a sufficient number of proteolytic peptides with highly accurate masses are required to make an unambiguous identification of a protein.18 To some extent, the use of either (7) Scheler, C.; Lamer, S.; Pan, Z. M.; Li, X. P.; Salnikow, J.; Jungblut, P. Electrophoresis 1998, 19, 918-927. (8) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390-9395. (9) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Gavik, B. M.; Yates, J. R. Nat. Biotechnol. 1999, 17, 676-682. (10) Haynes, P. A.; Yates, J. R. Yeast 2000, 17, 81-87. (11) Fenyo, D.; Qin, J.; Chait, B. T. Electrophoresis 1998, 19, 998-1005. (12) Neubauer, G.; King, A.; Rappsilber, J.; Calvio, C.; Watson, M.; Ajuh, P.; Sleeman, J.; Lamond, A.; Mann, M. Nat. Genet. 1998, 20, 46-50. (13) Jensen, O. N.; Podtelejnikov, A.; Mann, M. Rapid Commun. Mass Spectrom. 1996, 10, 1371-1378. (14) Jensen, O. N.; Podtelejnikov, A. V.; Mann, M. Anal. Chem. 1997, 69, 47414750. (15) Perkins, D. N.; Pappin, D. J., Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567. (16) Eriksson, J.; Chait, B. T.; Fenyo, D. Anal. Chem. 2000, 72, 999-1005. (17) Mann, M.; Hojrup, P.; Roepstorff, P. Biol. Mass. Spectrom. 1993, 22, 335338. (18) Clauser, K. R.; Baker, P.; Burlingame, A L. Anal. Chem. 1999, 71, 28712882. (19) Krause, E.; Wenschuh, H.; Jungblut, P. R. Anal. Chem. 1999, 71, 41604165.
Analytical Chemistry, Vol. 73, No. 20, October 15, 2001 4891
external or internal mass standards for calibrating peptide masses can increase the PMM mass accuracy. However, external calibrants typically give errors of 100 ppm or worse in individual matches and internal calibrants can result in signal suppression and signal overlap.20 These problems (low sequence coverage, mass degeneracy, chemical modification, calibration, etc.) substantially limit the specificity and accuracy of PMM database searches and lead to ambiguous results.7,16-20 Any information about a peptide sequence in a PMM can strengthen considerably the specificity of a database search.5,9,21-23 Although postsource decay (PSD) in MALDI-TOF MS can be used to determine the partial sequences of certain peptide ions,24 these experiments experience problems such as multiple steps, complexity of peptide fragmentation patterns, low yield of fragment ions, and the need for high mass accuracy.24-26 Individual peptide precursor ions can also undergo collision-induced dissociation (CID) through ESI-tandem MS (MS/MS) to generate the daughter spectra of peptide sequences.27-29 These “sequence tags” of particular peptides can be very specific in a database search for protein sequences derived from the genomic sequences.22,30-33 However, tandem MS/MS-based methods tend to be more complex and sample consuming than peptide mass mapping. The enormous number of putative proteins present in higher eukaryotes limits the use of the MS/MS strategy for protein identification on a large scale.30,34 Whereas CID MS/MS can become the ratelimiting step in the production of fragment ions for sequence analysis, large proteins, complexes, and mixtures require chromatographic separation to eliminate data compression and signal overlap for the unambiguous assignment of large numbers of proteolytic peptides from different proteins.21 In addition to data compression, ESI spectra can also suffer from poor resolution due to the multiple charge states of each species present in a peptide mixture.27 2D PAGE gels are used routinely to separate proteins according to their isoelectric points (IEF) and molecular weights (Mw).3 Although 2D PAGE is capable of providing high-resolution separation of proteins from cell extracts, it is tedious and time(20) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399. (21) Qin, J.; Fenyo, D.; Zhao Y.; Hall, W. W.; Chao, D. M.; Wilson, C. J.; Young, Z. R. A.; Chait, B. T. Anal. Chem. 1997, 69, 3995-4001. (22) Goodlett, D. R.; Bruce, J. E.; Anderson, G. A.; Rist, B.; Pasa-Tolic, L.; Fiehn, O.; Smith, R. D.; Aebersold, R. Anal. Chem. 2000, 72, 1112-1118. (23) Mo, W.; Ma, Y.; Takao, T.; Neubert, T. A. Rapid Commun. Mass Spectrom. 2000, 14, 2080-2081. (24) Chaurand, P.; Luetzenkirchen, F.; Spengler, B. J. Am. Soc. Mass Spectrom. 1999, 10, 91-103. (25) Kaufmann, R.; Kirsch, D.; Spengler, B. Rapid Commun. Mass Spectrom. 1996, 10, 1199-1208. (26) Keough, T.; Youngquist, R. S.; Lacey, M. P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136. (27) Hunt, D. F.; Yates, J. R.; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233-6237. (28) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-4057. (29) Qin, J.; Herring, C. J.; Zhang, X. Rapid Commun. Mass Spectrom. 1998, 12, 209-216. (30) Conrads, T. P.; Anderson, G. A.; Veenstra, T. D.; Pasa-Tolic, L.; Smith, R. D. Anal. Chem. 2000, 72, 3349-3354. (31) McCormack, A.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R. Anal. Chem. 1997, 69, 767-776. (32) Sechi, S.; Chait, B. T. Anal. Chem. 1998, 70, 5150-5158. (33) Egelhofer, V.; Bussow, K.; Luebbert, C.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 2741-2750. (34) Santoni, V.; Kieffer, S.; Desclaux, D.; Masson, F.; Rabilloud, T. Electrophoresis 2000, 21, 3329-3344.
4892
Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
consuming and its effectiveness is often reduced by a limited dynamic range and the exclusion of hydrophobic proteins.8,10 For example, membrane proteins often appear as trailing bands in the IEF dimension partly due to the noncovalent binding of lipids and proteins in the immobilized pH gradient strips.34,35 However, in the presence of detergents such as sodium dodecyl sulfate (SDS), hydrophobic proteins can remain soluble during separation under the denaturing conditions of a 1D SDS-PAGE gel. Although less time-consuming, 1D gel electrophoresis lacks resolution in the separation of complex mixtures and it is not uncommon for gel bands to contain two or more proteins. The presence of a large number of peptides produced by the proteolytic digestion of a band containing multiple protein species considerably restricts MS identification of proteins from a 1D gel band.9 Therefore, 1D SDS PAGE can only become a major separation scheme if multiple proteins in a 1D band can be identified simultaneously. Previously, we introduced a novel method for the site-specific mass tagging of cellular proteins with stable isotope-labeled amino acids during Escherichia coli culturing.36 In this report, we have extended this strategy to large-scale protein identification. In general, after generating a PMM of a protein mixture, MS can be used to recognize those peptides containing mass tags through their characteristic mass-split patterns. The magnitude of a mass split correlates directly with the partial amino acid composition of the mass-tagged peptide and can be accurately determined at more modest mass accuracy because the measurement of a mass difference in a mass split is relative and is independent of external or internal calibrations. Both m/z values and partial amino acid compositions of mass-tagged peptides can be used to constrain the database searches. In addition, the theoretical mass of a masstagged peptide can be used as an internal standard to recalibrate the entire peptide map. Consequently, the mass accuracy of a PMM with the mass-tag constraint can be substantially improved without the need for tandem MS/MS sequencing of individual peptides. As a result, we have unambiguously identified comigrating protein species in individual 1D SDS-PAGE bands. This allows a wider range of proteins, including membrane-bound and low-abundance proteins separated by 1D SDS-PAGE, to be efficiently identified by MS, thus reducing the need for 2D PAGE. Here, we demonstrate the identification of low-abundance proteins such as transcription factors, membrane proteins, and protein kinases from 1D SDS PAGE gels using mass-tag constrained PMMs. MATERIALS AND METHODS Chemicals. Stable isotope-enriched amino acid precursors, L-methionine-99.9%-d3 (Met-d3), D,L-serine-99.9%-d3 (Ser-d3), and L-tyrosine-99.9%-d2 (Tyr-d2) were purchased from Isotec INC. (Miamisburg, OH). Unlabeled amino acids, protease inhibitors, and R-cyano-4-hydroxycinnamic acid were obtained from Sigma (St. Louis, MO). Both dithiothreitol (DTT), and yeast nitrogen base (YNB) were from Fisher Scientific (Pittsburgh, PA). Sequencing-grade trypsin was purchased from Boehringer Man(35) Bell, A. W.; Ward, M. A.; Blackstock, W. P.; Freeman, H. N. M.; Choudhary, J. S.; Lewis, A. P.; Chotai, D.; Fazel, A.; Gushue, J. N.; Paiement, J.; Palcy, S.; Chevet, E.; Lafreniere-Roula, M.; Solari, R.; Thomas, D. Y.; Rowley, A.; Bergeron, J. J. M. J. Biol. Chem. 2001, 276, 5152-5165. (36) Chen, X.; Smith, L. M.; Bradbury, E. M. Anal. Chem. 2000, 72, 11341143.
Figure 1. (a) Experimental design for the generation, separation, and identification of amino acid-specific labeled (tagged) peptides from cellular protein complexes. The filled circle and symbol “X” designates the labeled amino acid (s). U, the unlabeled portion of the complex; L, the labeled portion of the complex. (b) Data searching design for the identification of proteins in a complex using peptide mass mapping constrained with stable isotope-tagged peptides.
nheim (Indianapolis, IN). Saccharomyces cerevisiae strain ATCC N442-4A (MATa his6 ade2 lys9 ura1 trp5 met2 arg4 mal suc) was obtained from the American Type Culture Collection (Manassas, VA). Residue-Specific Labeling of the Yeast Proteome. Yeast cells (N442-4A) were innoculated into 10 mL of synthetic complete (SC) medium37 and incubated overnight. The SC medium consists of 0.67% yeast nitrogen base containing all the essential vitamins, salts, and trace elements for cell growth, 0.5% ammonium sulfate, 2% dextrose, and a mixture of 20 amino acids. As shown in Figure 1a, the yeast cells from the overnight culture were diluted to a starting optical density (OD) of 0.1 in 100 mL of the SC medium supplemented with the labeled amino acid precursors, such as Met-d3 (200 mg/L), Ser-d3 (500 mg/L), or Tyr-d2 (500 mg/L). The labeled and unlabeled amino acid precursors were mixed in different ratios and added into the SC medium with other unlabeled amino acids to produce 0, 50, and 100% site-specifically labeled proteins, respectively. Cells were harvested during log phase (OD ∼ 1.0) and washed twice with millipure H2O (Millipore (37) Sherman, F. Methods Enzymol. 1991, 194, 3-21.
Corp., Bedford, MA) to remove excess medium. The cells were then resuspended in 10 mM Tris HCl (pH 8), 10 mM dithiothreitol, 10 mM ETDA, 100 mM NaCl, 0.1% SDS, and 5 mg/mL protease inhibitors including leupeptin, pepstatin A, and chemostatin A (Sigma). The cell lysate was prepared by vortexing with glass beads for 10 min at 4 °C followed by centrifugation at 14 000 rpm for 10 min at 4 °C to clarify the sample. One- and Two-Dimensional Separation of Yeast Proteins. Whole cell extracts were fractionated before loading on either 1D SDS-PAGE or 2D PAGE gels for protein separation. For 1D PAGE separation, the cell lysates (25 mg) were mixed with 6× SDS-PAGE sample buffer and heated at 90 °C for 5 min. The denatured samples were then separated using 8% polyacrylamide SDS gels and stained in the presence of Coomassie (G-250).38 In 2D PAGE separation, IEF utilized commercially available immobilized pH gradient (IPG) strips (pH 3-10, 18-cm length; BioRad Laboratories, Hercules, CA) with the protein IEF apparatus (Bio-Rad). Protein (100 µg) was mixed with IPG rehydration buffer (8 M urea, 2.5% CHAPS, 60 mM DTT, 0.2% Bio-Lytes) in a final volume of 200 µL. The IPG strips were allowed to rehydrate in Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
4893
Figure 2. MALDI-TOF peptide mass map of the tryptic digestion of a 2D PAGE gel spot isolated from yeast cell lysate labeled with Met-d3. The insets show expanded m/z scale for the peptide with the theoretical mass of 1740.8000 Da ((R)FDMSEFQEKHTVSR(L)) containing 0, 50, and 100% Met-d3, respectively.
the presence of protein samples overnight and focused at 70 000 V/h. After IEF, equilibrated strips were then apposed to the second dimension of SDS (13% C) gels, run at 40 mA for 6 h using the Protean II multicell gel apparatus (Bio-Rad), and then silverstained.38 Electroelution of Yeast Proteins from 1D Gel Slices and MALDI-TOF MS Analysis. Proteins were recovered from 1D SDS-PAGE gel slices by electroelution. Electroelution was performed using a model 422 Bio-Rad electroelution apparatus in a buffer containing 50 mM ammonium bicarbonate (pH7.5), 0.1% SDS and a constant current of 15 mA for 4 h. The eluted proteins were concentrated by precipitation with an equal volume of 10% cold trichloroacetic acid (TCA) on ice for 1 h followed by centrifugation at 14 000 rpm. The protein precipitates were desalted for MALDI-TOF analysis using C4 ZipTips (Millipore Corp.). The protein was eluted from the resin using 60% acetonitrile, 0.1% trifluoracetic acid (TFA). Samples were then diluted 9:1 with sinipinic acid (Sigma), 10 mg/mL in 0.1% TFA. Tryptic Digestion, Peptide Extraction, and MS Analysis. Gel bands or spots of interest were sliced from 1D or 2D gels. The Coomassie-stained gel slices were destained with a acetonitrile and 50 mM NH4HCO3 at 1:1 ratio. The silver-stained 2D gel (38) Merril, R. C. Methods Enzymol. 1990, 182, 477-488.
4894 Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
slices were destained using a 30 mM potassium fericyanide and 100 mM sodium thiosulfate at 1:1 ratio. Destained gel slices were then dried using a speed-vacuum centrifuge. Trypsin (Boehringer Mannheim) was added in a final concentration of 10 µg/mL, and the mixture was incubated overnight or 12 h at 37 °C. The tryptic peptides were extracted from gel slices in two steps. First, the gel slices were suspended in 5% acetic acid solution and sonicated at 37 °C for 45 min. The supernatant was reserved in a fresh microcentrifuge tube on ice. The gel slices then continue to be extracted with 50% acetonitrle/5% acetic acid for an additional 45 min. Both supernatants were combined and lyophilized. The samples were then resuspended in a 0.1% TFA solution and further desalted using C18 ZipTips. Peptides were eluted from the C18 resin stepwise with 0.1% TFA solution containing 10, 30, and 60% acetonitrile, respectively. For mass spectrometric analysis, 1 µL of sample was mixed with 19 µL of a saturated solution (10 mg/mL) of R-cyano-4hydroxycinnamic acid (Sigma), which was prepared by dissolving 10 mg in 1 mL of a 1:1 solution of acetonitrile and 0.1% TFA. All mass spectrometry experiments were carried out on a PE Voyager DE-STR Biospectrometry workstation equipped with a N2 laser (337 nm, 3-ns pulse width, 20-Hz repetition rate) in both linear and reflectron modes (PE Biosystems, Framingham, MA). The
Table 1. Yeast Proteins Identified from 2D PAGE Spots Using Peptide Mass Mapping Constrained with Met-d3-Tagged Peptide(s)
proteina
Mw
Sec7p
205
pI
seq and cal m/z (Da) of Met-d3 peptide(s)
obs m/z (Da) of Met-d3-peptide(s)
matched untagged peptidesb (Da) ac bd
4.66
(R)KMTQNVADICFYNENL(T) 2004.9159
2005.0941
1609.0776 2088.3659 2771.0162 1141.9445 1522.5756 2041.4298 2165.4979 1046.7003 1570.8405 2002.1800 2273.4113 1068.9831 1159.9629 1579.3314 2039.7904 2124.8065 2584.2245
Hsp78p
91.3
6.13
(R)FDMSEFQEKHTVSR(L) 1740.8000
1740.3341
Ade13p
54.5
6.2
(K)EVEEPFEKSQIGSSAMAYK(R) 2146.0014
2146.2600
Eno2p
46.9
5.82
(K)LGANAILGVSMAAAR(A) 1414.7840
1415.2406
PGK
44.7
7.4
1444.5993 1460.6925
Adh1p
36.8
6.45
(R)AHSSMVGFDLPQR(A) 1444.7007 1460.6925 (R)YVRANGTTVLVGMPAGAK(C) 968.4776
Hem13p
37.7
6.57
(R)NLPIRQQMEALIRR(K) 2034.0694
2034.6650
968.4830
1050.4612 1119.4005 1439.7513 1251.6697 1564.9480 1804.0710
1615.4345 1632.4379 1760.5636
728.4213 1609.8432 2088.0250 2771.4275 1141.6549 1522.7681 2042.0995 2166.1268 1046.5020 1570.6075 2002.0023 2273.3542 1068.6383 1159.5886 1578.8218 1855.0374 1876.1519 2039.1323 2124.1209 2583.3907 1439.8111 1768.0321 2327.1268 1017.5408 1251.6697 1411.7418 1618.8969 2270.3191 2312.2943 972.4536 1614.8662 1631.8635 1759.9442
sequence coverage (%) a b 4
4
7
7
16
16
26
33
10
15
15
27
9
13
a Proteins were named as found in the Yeast Proteome Database (YPD).41,42 bProteins were identified using MS-FIT search18 in NCBInR database that were constrained by the following parameters: PI, Mw, species, and up to three missed tryptic cleavage sites. cPeptide mass maps were calibrated using calmix1. dPeptide masses were recalibrated using the theoretical masses of the mass-tagged peptides from column 4.
mass spectra of the tryptic digests were acquired in the reflectron mode with delayed extraction. The m/z values of proteolytic peptides were calibrated with Calmix 1 (PE Biosystems). Postsource decay fragment ion spectra were acquired for those peptides containing the labeled amino acids after isolation of the desired precursor ion. Fragment ions were refocused onto the detector by stepping the voltage applied to the reflectron in a series of mirror ratios.36 Protein Identification Using PMM Constrained by the Mass Tagging of Peptides. As illustrated in Figure 1b, in a PMM, those peptides tagged with labeled amino acid(s) can be distinguished from other peptides by their characteristic mass-split patterns. The partial amino acid composition of a mass-tagged peptide is determined by the difference between the labeled and unlabeled peaks in a mass-split pattern. Proteolytic peptides derived from the theoretical digestion of various proteins translated from the genomic sequence or EST databases are filtered with the content of the labeled amino acid residue(s) resulting in a mass-tag constrained proteolytic peptide library. The database search for the proteins corresponding to the mass-tagged peptides is constrained by two parameters, the measured m/z values and the content of the labeled amino acids, which allows for a far more selective and confident protein sequence search than by peptide masses alone. The theoretical m/z values of those mass-tagged peptides with the highest confidence scores in the first-round two-
parameter search can then be used as internal calibrants for other peptide masses in the same peptide map. After the recalibration against the tagged peptides, a second-round data search is performed with the newly calibrated m/z values of individual peptides. RESULTS Site-Specific Mass Tagging of the Yeast Proteome with Stable Isotope-Labeled Amino Acids. Using our labeling strategy (Figure 1), three labeled precursors, methionine-S-methyld3 (Met-d3), tyrosine-3,3-d2 (Tyr-d2), and serine-2, 3,3-d3 (Ser-d3), were incorporated into the S. cerevisiae proteome. On the basis of their deuterium contents, each Met-d3 or Ser-d3 residue contains a 3-Da mass tag whereas a Tyr-d2 has a 2-Da mass tag. These mass-tagged precursors are used as internal signatures for certain proteolytic peptides containing these residues to characterize the parent proteins. Figure 2 shows a MALDI-TOF PMM of the tryptic digests of a silver-stained 2D gel spot isolated from the Met-d3labeled cell extracts. Among the tryptic peptides detected, there is a peptide peak with an m/z value of 1740.3341 Da (M+ ion) showing a 3-Da mass shift when compared with the same peptide isolated from yeast cells grown in the presence of 50 and 100% Met-d3, respectively (inset, Figure 2). Through the characteristic 3-Da mass-split pattern, this Met-containing peptide has been identified in the tryptic peptide map and its theoretical mass has Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
4895
Figure 3. MALDI-TOF mass spectra of an in-gel tryptic digest of 50-kDa band labeled with 0, 50, or 100% Met-d3 isolated from 1D SDSPAGE gel. (a) The peptide mass map with external calibration; (b) the peptide mass map after recalibration using a Met-tagged peptide and its m/z value. The peptides have been assigned to Eno1p (red), GATA-1p (green), and ADHIIIp (blue), respectively. Insets shows expanded views of Met-d3-tagged peptides with theoretical masses of 1430.7500 and 1789.8444 Da containing 0, 50, and 100% Met-d3, respectively.
been determined as 1740.8000 Da. This assignment was verified by the increasing intensity of the labeled peaks with increasing percentage of Met-d3 in the SC media. No isotopic scrambling was observed for these peptides without methionine residues. High Specificity of PMMs Constrained by Mass-Tagged Peptides in Protein Identification. The S. cerevisiae genome contains a total of 6118 possible open reading frames (ORFs).22 Theoretically, a complete tryptic digestion of the 6118 possible ORFs would be expected to generate 344 855 peptides.22 The number of matches for a tryptic peptide depends on the error tolerance in the database search. For example, using the m/z value of 1740.8000 Da, a search of the theoretical digest pool identifies 475 peptide isobars at an error tolerance of 500 ppm and 21 at an error of 250 ppm. However, including the methionine content of the peptide in the search reduces significantly the number of possible peptides containing a single methionine to 120 at 500 ppm and 4 at 250 ppm. Further, the apparent Mw of the protein in the gel spot limits the number of possible peptides defined by both m/z values and their Met tags. To demonstrate the effectiveness of our strategy, we have selected at random seven protein spots in the Mw range of 30 000-200 000 from a silver-stained 2D PAGE gel as summarized in Table 1. Following the method 4896 Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
as described in Figure 1b, the search for Met-containing peptide(s) of particular m/z value(s) using MS-FIT18 in the NCBInR database was first performed in the error tolerance range of 200500 ppm. After each PMM was further constrained by one or two mass-tagged peptides, a relatively low sequence coverage for each protein (
