High-Throughput Comparative Proteome Analysis Using a

---

Anal. Chem. 2004, 76, 5345-5353

High-Throughput Comparative Proteome Analysis Using a Quantitative Cysteinyl-peptide Enrichment Technology Tao Liu,† Wei-Jun Qian,† Eric F. Strittmatter, David G. Camp, II, Gordon A. Anderson, Brian D. Thrall, and Richard D. Smith*

Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352

A new quantitative cysteinyl-peptide enrichment technology (QCET) was developed to achieve higher efficiency, greater dynamic range, and higher throughput in quantitative proteomics that use stable-isotope labeling techniques combined with high-resolution liquid chromatography (LC)-mass spectrometry (MS). This approach involves 18O labeling of tryptic peptides, high-efficiency enrichment of cysteine-containing peptides, and confident protein identification and quantification using the accurate mass and time tag strategy. Proteome profiling of naı1ve and in vitro-differentiated human mammary epithelial cells using QCET resulted in the identification and quantification of 603 proteins in a single LC-Fourier transform ion cyclotron resonance MS analysis. Advantages of this technology include the following: (1) a simple, highly efficient method for enriching cysteinyl-peptides; (2) a high-throughput strategy suitable for extensive proteome analysis; and (3) improved labeling efficiency for better quantitative measurements. This technology enhances both the functional analysis of biological systems and the detection of potential clinical biomarkers. Quantitative proteomic measurements are important in studies aimed at discovering disease biomarkers and providing new insights into biological processes and disorders. However, comprehensive proteome analysis remains technically challenging due to issues associated with both sample complexity and the wide dynamic range of protein abundances. The strategy of combining stable-isotope labeling with automated liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) is increasingly being applied in quantitative proteomic studies.1-5 Automated LC-MS/MS provides high sensitivity and proteome coverage, as well as accurate relative quantitation. Stable isotopes can generally be introduced into proteins or peptides chemically,1,2 metaboli† Authors who contributed equally to this work. (1) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (2) Zhou, H. L.; Ranish, J. A.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2002, 19, 512-515. (3) Veenstra, T. D.; Martinovic, S.; Anderson, G. A.; Pasa-Tolic, L.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2000, 11, 78-82. (4) Ong, S. E.; Kratchmarova, I.; Mann, M. J. Proteome Res. 2003, 2, 173-181. (5) Yao, X. D.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-2842.

10.1021/ac049485q CCC: $27.50 Published on Web 08/05/2004

© 2004 American Chemical Society

cally,3,4 or enzymatically,5 and different mass shifts can be generated by using various labeling reagents. To attain in-depth analysis of low-abundance proteins in complex mixtures, additional methods are required to reduce sample complexity. One such method involves initially isolating the functional multiprotein complexes or organelles from the cells prior to analysis.6,7 Another approach involves selectively enriching a subset of peptides from the complex mixture. This has been most commonly achieved by targeting peptides that contain amino acids with specific functional groups, such as chemically reactive cysteinyl (Cys) residues1,2,7,8 or residues that have been posttranslationally modified with a phosphate9 or carbohydrate.10 A further reduction in sample complexity can be achieved by using multidimensional separations prior to MS analyses.11 Isotope-coded affinity tags (ICAT) approach is one of the most widely applied labeling techniques used in quantitative proteomic studies. ICAT employs an isotopically distinct region flanked by iodoacetamide and biotin functionalities that allow for the modification and extraction of reduced Cys-containing peptides (Cyspeptides) using immobilized avidin chromatography.1 The firstgeneration ICAT reagents caused differential LC elution of tagged peptide pairs that resulted in variable ionization efficiencies for the two peptides, thereby affecting quantitation.12 This effect can be further compounded by limitations associated with avidin affinity enrichment, such as residual nonspecifically bound peptides from the affinity matrix and reduced sample recovery due to irreversible binding of a subpopulation of the biotinylated peptides. New version ICAT reagents have been developed to resolve the retention time issue, reduce the tag size,13-15 and utilize a solid-phase approach.2 (6) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R. Nat. Biotechnol. 1999, 17, 676-682. (7) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nat. Biotechnol. 2001, 19, 946951. (8) Gevaert, K.; Ghesquie`re, B.; Staes, A.; Martens, L.; Van Damme, J.; Thomas, G. R.; Vandekerckhove, J. Proteomics, in press. (9) Oda, Y.; Nagasu, T.; Chait, B. Nat. Biotechnol. 2001, 19, 379-382. (10) Zhang, H.; Li, X. J.; Martin, D. B.; Aerbersold, R. Nat. Biotechnol. 2003, 21, 660-666. (11) Washburn, M. P.; Ulaszek, R.; Deciu, C.; Schieltz, D.; Yates, J. R. Anal. Chem. 2002, 74, 1650-1657. (12) Zhang, R.; Regnier, F. E. J. Proteome Res. 2002, 1, 139-147. (13) Yu, L. R.; Conrads, T. P.; Uo, T.; Issaq, H. J.; Morrison, R. S.; Veenstra, T. D. J. Proteome Res., in press.

Analytical Chemistry, Vol. 76, No. 18, September 15, 2004 5345

Here we describe a new quantitative cysteinyl-peptide enrichment technology (QCET) that enables higher efficiency, greater dynamic range, and higher throughput quantitative proteomic analyses than previous labeling technologies. QCET is based on stable-isotope labeling of global tryptic peptides by trypsincatalyzed 16O-to-18O exchange,16,17 high-efficiency enrichment of Cys-peptides using a thiol-specific covalent resin, and the accurate mass and time (AMT) tag approach.18-20 QCET was evaluated using both model mixtures of either peptides or proteins and then used to systematically identify and determine the ratios of protein abundance in human mammary epithelial cells (HMEC) with and without treatment by phorbol 12-myristate 13-acetate (PMA). HMEC cell line is a well-characterized model cell line suitable for investigating growth factor regulatory cascades and for comparative studies involving mammary cancer cells. Exposure of HMEC to PMA, a tumor promoting agent, stimulates a cascade of events associated with differentiation to a basal epithelial cell phenotype, including activation of protein kinase C, secretion and shedding of cell surface proteins, and transactivation of epidermal growth factor receptor signaling.21,22 EXPERIMENTAL SECTION Preparation of HMEC Protein Digests. Nontumorigenic HMEC strain 184 A1L523 was used; cells were routinely cultured in DFCI-1 medium (Gibco BRL, Gaithersburg, MD) as previously described24 until 90% confluence was achieved. The use of HMEC cells was reviewed by the Pacific Northwest National Laboratory IRB for human subjects research in accordance with federal regulations. Cells in eight dishes (1 × 107 cells/dish) were treated with 200 nM PMA for 24 h while cells in another eight dishes were cultured under normal conditions. Cell pellets were washed three times with ice-cold phosphate-buffered saline. Lysis buffer (10 mM Tris, 150 mM NaCl, 1% NP-40, 1 mM NaVO3, 10 mM NaF, and protease inhibitor cocktail, pH 7.4) was added to the cell pellets, and the cells were lysed using intermittent sonication on ice. The lysates were centrifuged for 20 min at 4 °C at 14000g to pellet any cell debris, and then 100 µg of proteins from each of the two lysates was digested separately as follows. Proteins were denatured and reduced in 50 mM Tris buffer, pH 8.2, 8 M urea, 10 mM tributyl phosphine for 1 h at 37 °C. The protein sample was diluted 10 times using 20 mM Tris buffer (pH 8.2) and digested overnight at 37 °C using sequencing grade, modified (14) Li, J.; Steen, H.; Gygi, S. P. Mol. Cell. Proteomics 2003, 2, 299-314. (15) Hansen, K. C.; Schmitt-Ulms, G.; Chalkley, R. J.; Hirsch, J.; Baldwin, M. A.; Burlingame, A. L. Mol. Cell. Proteomics 2003, 2, 299-314. (16) Yao, X.; Afonso, C.; Fenselau, C. J. Proteome Res. 2003, 2, 147-152. (17) Heller, M.; Mattou, H.; Menzel, C.; Yao, X. J. Am. Soc. Mass Spectrom. 2003, 14, 704-718. (18) Smith, R. D.; Anderson, G. A.; Lipton, M. S.; Pasa-Tolic, L.; Shen, Y.; Conrads, T. P.; Veenstra, T. D.; Udseth, H. R. Proteomics 2002, 2, 513-523. (19) Strittmatter, E. F.; Ferguson, P. L.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2003, 14, 980-991. (20) Lipton, M. S.; Pasa-Tolic, L.; Anderson, G. A.; Anderson, D. J.; Auberry, D. L.; Battista, J. R.; Daly, M. J.; Fredrickson, J.; Hixson, K. K.; Kostandarithes, H.; Masselon, C.; Markillie, L. M.; Moore, R. J.; Romine, M. F.; Shen, Y.; Stritmatter, E.; Tolic, N.; Udseth, H. R.; Venkateswaran, A.; Wong, K. K.; Zhao, R.; Smith, R. D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049-11054. (21) Grunberg, E.; Eckert, K.; Karsten, U.; Maurer, H. R. Tumor Biol. 2000, 21, 211-223. (22) Dong, J.; Wiley, H. S. J. Biol. Chem. 2000, 275, 557-565. (23) Stampfer, M. R.; Yaswen, P. Cancer Surv. 1993, 18, 7-34. (24) Chen, W.-N. U.; Woodbury, R. L.; Kathmann, L. E.; Opresko, L. K.; Zangar, R. C.; Wiley, H. S.; Thrall, B. D. J. Biol. Chem. 2004, 279, 18488-18496.

5346

Analytical Chemistry, Vol. 76, No. 18, September 15, 2004

porcine trypsin (Promega, Madison, WI) at a trypsin/protein ratio of 1:50. The digests were purified using SPE C18 column (Supelco, Bellefonte, PA) and dried under vacuum. Preparation of Standard Mixture Digests. Two mixtures, each containing the following five proteins but at different concentrations (pmol/µL mixture A, pmol/µL mixture B) were obtained from Sigma (St. Louis, MO): bovine serum albumin (BSA) (15, 15); bovine ribonuclease A (15, 90); chicken lysozyme (90, 15); chicken ovalbumin (45, 15); and rabbit glyceraldehyde3-phosphate dehydrogenase (15, 45). A total of 100 µg of each mixture was reduced, digested, desalted, and dried as described above. Trypsin-Catalyzed 18O Labeling. To dissolve the dried peptides, 20 µL of acetonitrile was first added to the dried digest, followed by the addition of 100 µL of 50 mM NH4HCO3 in either 18O-enriched water (95%, Isotec, Miamisburg, OH) or regular 16O water. Then, 1 µL of 1 M CaCl2 and 5 µL of immobilized trypsin (Applied Biosystems, Foster City, CA) were added to the digests, and the samples were mixed continuously for 24 h at 30 °C. For the HMEC samples, the control sample was labeled with 16O and the PMA-treated sample, with 18O. For the standard protein mixtures, A was labeled with 16O and B, with 18O. Supernatants were collected by centrifuging the samples for 5 min at 15000g. The corresponding 16O- and 18O-labeled samples were pooled, combined, and then dried under vacuum. Cys-peptide Covalent Enrichment by Thiopropyl Sepharose 6B. The combined 16O/18O-labeled sample was dissolved in 100 µL of 50 mM Tris buffer, pH 7.5, 21 mM EDTA (coupling buffer). A total of 100 µL of Thiopropyl Sepharose 6B affinity resin (Amersham Biosciences, Uppsala, Sweden) was prepared from dried powder per the manufacturer’s instructions. The sample was incubated with the resin for 1 h at 25 °C with gentle mixing. The resin was then washed sequentially with 2 M NaCl, 80% acetonitrile/0.1% TFA solution, and 50 mM Tris buffer. To release the bound Cys-peptides, 100 µL of 20 mM dithiothreitol (DTT) in 50 mM Tris buffer was added to the resin and incubated for 30 min at 25 °C. The sample was alkylated with iodoacetamide, desalted by SPE C18 column, and dried under vacuum. A 50-µL aliquot of 25 mM NH4HCO3 was used to dissolve the samples, and 10 µL of each sample was used for LC-MS/MS analysis. Prior to LCFourier transform ion cyclotron resonance (FTICR) analysis, the samples were diluted 5-fold, and 10 µL of each diluted solution was consumed during analysis. Yield Determination of Thiol-Specific Capture and Release. A sample consisting of 20 nmol of Cys-containing somatostatin peptide (sequence GCKNFFWK; Sigma) and 10 nmol of non-Cys-containing anaphylatoxin C3a peptide (sequence ASHLGLAR; Sigma) were dissolved in 20 µL of coupling buffer and incubated with 20 µL of Thiopropyl Sepharose 6B resin for 1 h at 25 °C with gentle mixing. The unbound portion was removed from the resin and saved as a reference. After washing, the Cyspeptide was released by incubating the resin with 20 µL of 20 mM DTT in 50 mM Tris buffer for 30 min. A total of 10 nmol of anaphylatoxin C3a peptide was spiked into the eluate as a standard. The eluate, the unbound portion, and the original peptide mixture were all diluted to 600 µL by 25 mM NH4HCO3, and 10 µL of each diluted solution was analyzed by LC-MS.

Strong Cation Exchange (SCX) Fractionation of Enriched Cys-peptides. Cys-peptides enriched from the HMEC tryptic digest were reconstituted in 1.0 mL of a 10 mM ammonium formate, 25% acetonitrile, pH 3.0 solution and injected for SCX separation onto a Polysulfoethyl A 200 × 9.4 mm column (PolyLC, Columbia, MD) with a flow rate of 1 mL/min. The separations were performed with a LC-10A system (Shimadzu, Columbia, MD) with mobile phases consisting of 10 mM ammonium formate, 25% acetonitrile, pH 3.0 (A), and 500 mM ammonium formate, 25% acetonitrile, pH 6.8 (B). Once loaded, the run was isocratic for 10 min at 100% A. Peptides were then separated by a gradient from 0 to 50% B over 40 min, followed by a gradient of 50-100% B over 10 min. A total of 35 fractions was collected, after which each fraction was lyophilized to dryness and stored at -80 °C until analyzed. Capillary LC-MS/MS and LC-FTICR Analysis. Peptide samples were analyzed using a custom-built capillary LC system25 coupled on-line to either a LCQ Deca XP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) or an Apex III 9.4-T FTICR mass spectrometer (Bruker Doltonics, Billerica, MA) using an in-house-manufactured ESI interface. The reversed-phase capillary column was made by slurry packing 5-µm Jupiter C18 bonded particles (Phenomenex, Torrence, CA) into a 65-cm-long, 150-µmi.d. fused-silica capillary (Polymicro Technologies, Phoenix, AZ). The mobile phase consisted of 0.2% acetic acid and 0.05% TFA in water (A) and 0.1% TFA in 90% acetonitrile/10% water (B). Mobile phases were degassed on-line using a vacuum degasser (Jones Chromatography Inc., Lakewood, CO). After injecting 10 µL of peptide sample onto the reversed-phase capillary column, the mobile phase was held at 100% A for 20 min. Exponential gradient elution was performed by increasing the mobile-phase composition to ∼70% B over 150 min using a stainless steel mixing chamber. The same gradient was applied for LC-MS/MS and LC-FTICR analysis. The ion trap and FTICR mass spectrometers were operated as described previously.18,26 Data Analysis. For LC-MS/MS analyses, peptides were identified by searching the MS/MS spectra against databases that contained either the sequences of the five model proteins or a nonredundant human International Protein Index (IPI) database (consisting of 47 306 protein entries at the time of our analysis; available on-line at http://www.ebi.ac.uk/IPI) using SEQUEST27 (Thermo Finnigan). The criteria used to filter raw SEQUEST identifications were similar to that reported by Washburn et al.28 Briefly, a ∆ correlation (∆Cn) value higher than 0.1 and crosscorrelation score (Xcorr) cutoff values of 1.9, 2.2, and 3.75 were used for tryptic peptides with charge states of 1+, 2+, and 3+, respectively. Nontryptic peptides were not considered. The peptide retention times from each LC-MS/MS analysis were normalized to a range of 0-1 using a procedure similar to that previously reported29 with the exception of using a quadratic instead of linear (25) Shen, Y.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K.; Pasa-Tolic, L.; Veenstra, T. D.; Lipton, M. S.; Smith, R. D. Anal. Chem. 2001, 73, 1766-1775. (26) Belov, M. E.; Anderson, G. A.; Wingerd, M. A.; Udseth, H. R.; Tang, K.; Prior, D. C.; Swanson, K. R.; Buschbach, M. A.; Strittmatter, E. F.; Moore, R. J.; Smith, R. D. J. Am. Soc. Mass. Spectrom. 2004, 15, 212-232. (27) Eng, J. K.; McCormack, A. L.; Yates, J. R. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989. (28) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2001, 19, 242247.

Figure 1. Strategy for quantification of differential protein expression. Two protein mixtures representing two different cell states are digested by trypsin separately. The resulting tryptic peptides are labeled by trypsin-catalyzed oxygen exchange using 16O- and 18Oenriched water, respectively. The two samples are combined, and cysteinyl-peptides are selectively captured and released using thiolaffinity resin. The enriched cysteinyl peptides are first analyzed by LC-MS/MS generating a PMT tag database that includes the calculated mass and normalized elution time for each identified peptide. The same peptide sample is analyzed by LC-FTICR, and peptides are identified and quantified as AMT tags by matching to the PMT tag database without the need for additional MS/MS analyses. Once a PMT tag database is established for a biological system, the system can be extensively investigated in a highthroughput manner by analyzing samples generated under different conditions using LC-FTICR.

equation to obtain normalized elution times (NETs). Both the calculated mass and NET of the identified peptides were included in a potential mass and time (PMT) tag database. Peptides/ proteins were identified by validating PMT tags in the database as AMT tags and quantified using in-house-developed VIPER software. A maximum mass error of 5 ppm and a maximum NET error of 5% were used for searching the PMT tag database. A factor of 1.10 was used to adjust the observed abundance ratios since the final percentage of 18O water in our protocol is ∼90%. RESULTS AND DISCUSSION QCET Principles. QCET involves several sequential steps (Figure 1). First, protein samples from two cell states are prepared, separately digested by trypsin under identical conditions, and the tryptic peptides from each sample are labeled with either 16O or 18O by immobilized-trypsin-catalyzed oxygen exchange in either regular or 18O-enriched water. Next, the differentially labeled peptide samples are combined and Cys-peptides are selectively captured using a thiol-specific affinity resin by forming a disulfide bond between the Cys-peptide and the resin (Figure 2A). Following stringent washing, bound Cys-peptides are released from the resin by incubating with DTT. Finally, the enriched Cys-peptides are analyzed, identified, and quantified using the AMT tag approach.18 (29) Petritis, K.; Kangas, L. J.; Ferguson, P. L.; Anderson, G. A.; Pasa-Tolic’, L.; Lipton, M. S.; Auberry, K. J.; Strittmatter, E.; Shen, Y.; Zhao, R.; Smith, R. D. Anal. Chem. 2003, 75, 1039-1048.

Analytical Chemistry, Vol. 76, No. 18, September 15, 2004

5347

Figure 2. Principle of cysteinyl-peptide enrichment method. (A) Reaction scheme for covalent chromatography of a cysteinyl-peptide (R-SH) on Thiopropyl Sepharose 6B. R′-SH represents a low molecular weight thiol such as DTT. (B) Utilization of LC-MS analyses of a peptide mixture consisting of a cysteine-containing somatostatin peptide (*) and a non-cysteine-containing anaphylatoxin C3a peptide (#) to validate the efficiency of cysteinyl-peptide capture and release. Top panel shows the ion chromatogram of somatostatin peptide (/, m/z ) 515.50, 2+ ion) and anaphylatoxin C3a peptide (#, m/z ) 412.90, 2+ ion) before binding to the resin. Middle panel shows ion chromatogram of the solutes remaining after 1-h capture, indicating virtually complete disappearance of somatostatin peptide signal. The bound cysteinyl-peptide was released from the resin by incubating the resin for 1 h with DTT. Bottom panel shows that the somatostatin peptide (*) was quantitatively recovered. Identical amounts of anaphylatoxin C3a peptide were presented in original and released samples for comparison. 5348 Analytical Chemistry, Vol. 76, No. 18, September 15, 2004

Proteolytic 18O labeling of proteins has been previously applied in various proteomic studies.5,30 Unlike deuterium, 18O incurs a minimal and generally negligible isotopic effect on chromatographic elution. Thus, 16O/18O-labeled peptide pairs coelute, minimizing errors potentially introduced by differences in electrospray ionization suppression effects. For QCET, a postdigestion 16O-to-18O exchange reaction was employed, catalyzed by immobilized trypsin, to exclusively label the C-termini of tryptic peptides with two 18O atoms, creating a mass shift of 4 Da between the unlabeled and the 18O-labeled peptides. By using this postdigestion 18O labeling strategy, reagents introduced during the digestion step (e.g., urea, DTT, etc.) can be effectively removed before labeling the tryptic peptides. The control experiments using our optimized protocol for labeling BSA showed that 97.5% peptides were labeled with two 18O atoms (data not shown). In addition, only those peptides with Lys or Arg at their C-termini are labeled using the postdigestion 16O-to-18O exchange reaction, which suggests high enzyme specificity of the labeling (data not shown). The thiol-covalent capture/release reaction, which has been applied to enrich chemically modified glycopeptides and phosphopeptides,31,32 also has the potential for high-efficiency enrichment of Cys-peptides at the proteome level. The thiol-affinity resin reacts with reduced sulfhydryl groups to yield a mixed disulfide and 2-thiopyridone. Stringent washing after Cys-peptide capture results in efficient removal of nonspecifically bound peptides. The covalently captured Cys-peptides are subsequently released from the resin by addition of a reducing agent (e.g., DTT; see Figure 2A). The entire peptide capture/wash/release process is complete within 2 h. In this work, a model peptide mixture consisting of somatostatin (GCKNFFWK) and non-cysteine-containing anaphylatoxin C3a (ASHLGLAR) (Figure 2B) was used to illustrate the efficiency of the capture/release reactions. The somatostatin was completely captured by the resin after 1 h of incubation (compare top and middle panels of Figure 2B). Following a 30-min incubation of the resin with DTT, the bound somatostatin was quantitatively recovered (compare top and bottom panels of Figure 2B), indicating the high specificity and efficiency of the capture/release reactions. For complex proteomic samples, all Cys-peptides were alkylated, following enrichment, to increase the MS detection efficiency. The high-throughput AMT tag portion of QCET involves two stages. In the first stage, a PMT tag database containing calculated mass and LC elution time information for each peptide is generated on the basis of confident LC-MS/MS peptide identifications. In the second stage, peptide samples from the same proteome are analyzed by high mass accuracy FTICR mass spectrometry under identical LC conditions. When both the measured masses and elution times of FTICR-detected peptides match the PMT tags within a particular error window, the PMT tags are validated as AMT tags, bypassing additional LC-MS/ MS analyses and allowing extensive high-throughput investigation of a biological system. Peptide elution times are normalized into a range of 0-1 to enable accurate comparison among LC-MS (30) Wang, Y. K.; Ma, Z.; Quinn, D. F.; Fu, E. W. Anal. Chem. 2001, 73, 37423750. (31) Wells, L.; Vosseller, K.; Cole, R. N.; Cronshaw, J. M.; Matunis, M. J.; Hart, G. W. Mol. Cell. Proteomics 2002, 1, 791-804. (32) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826-6836.

Table 1. Quantitative Analysis of Protein Mixture Components gene namea

peptide sequence identifiedb

observed ratioc (16O/18O)

ALBU•BOVIN

CCAADDK CCTESLVNR DAIPENLPPLTADFAEDKDVCK DDPHACYSTVFDKLK EACFAVEGPK ECCDKPLLEK ECCHGDLLECADDR EYEATLEECCAK GACLLPK GLVLIAFSQYLQQCPFDEHVK LCVLHEK LFTFHADICTLPDTEK LKPDPNTLCDEFK MPCTEDYLSLILNR NECFLSHK QNCDQFEK RPCFSALTPDETYVPK SHCIAEVEK SLHTLFGDELCK TCVADESHAGCEK YICDNQDTISSK YNGVFQECCQAEDK ADHPFLFCIK CFDVFK LPGFGDSIEAQCGTSVNVHSSLR YPILPEYLQCVK IVSNASCTTNCLAPLAK VPTPNVSVVDLTCR CELAAAMK CKGTDVQAWIR GYSLGNWVCAAK NLCNIPCSALLSSDITASVNCAK WWCNDGR CKPVNTFVHESLADVQAVCSQK NGQTNCYQSYSTMSITDCR QHMDSSTSAASSSNYCNQMMK YPNCAYK

0.95 0.95 1.09 0.86 1.02 0.98 1.10 0.85 1.11 1.14 1.05 1.10 1.07 1.12 0.96 0.85 0.97 1.13 1.09 1.07 1.01 1.11 2.94 3.16 3.19 2.98 0.32 0.37 5.98 6.44 6.52 6.59 6.40 0.23 0.19 0.18 0.17

OVAL•CHICK

G3P•RABIT LYC•CHICK

RNP•BOVIN

mean ( SD

expected ratiod (16O/18O)

% error

1.03 ( 0.09

1.00

3.0

3.07 ( 0.13

3.00

2.3

0.35 ( 0.04

0.33

6.1

6.39 ( 0.24

6.00

6.5

0.19 ( 0.03

0.17

11.8

a Gene names are according to Swiss-Prot nomenclature. b All cysteine residues were alkylated by iodoacetamide. c All observed ratios were divided by a factor of 1.10 (See Experimental Section for details). d Expected ratios were calculated from the known amounts of proteins present in each mixture.

data sets,29 and these NET values are highly reproducible (variance 95% of the observed peptide pairs were identified as unique AMT tags. (4) The highly efficient postdigestion 18O labeling strategy incorporates two atoms of 18O in almost all tryptic peptides, providing the framework for accurate quantitation. In QCET, the stable-isotope labeling is at the peptide level, but we note that the combination of Cys-peptide enrichment and high-throughput AMT tag approach can also be coupled to other labeling methods where the labeling is at the protein level, such as 15N metabolic labeling3 and labeling using amino acids in cell culture.4 The ease and efficiency of enrichment, accuracy of

quantitation, flexibility of coupling to different isotopic labeling methods, and high-throughput potential of QCET should enable broad applications. ACKNOWLEDGMENT We thank the NIH National Center for Research Resources (RR018522) and the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy’s (DOE) Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL), for supporting portions of this research. PNNL is operated by Battelle for the DOE under Contract DEAC06-76RLO 1830. SUPPORTING INFORMATION AVAILABLE A table of the 603 proteins identified and quantified listing their identity, database access code, and 16O/18O ratio between the naı¨ve and PMA-treated cells. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 2, 2004. Accepted June 23, 2004. AC049485Q

Analytical Chemistry, Vol. 76, No. 18, September 15, 2004

5353