High-Throughput Peptide Identification from Protein Digests Using

Loïc Quinton, Jean-Pierre Le Caer, Gilles Phan, Caroline Ligny-Lemaire, Julie ... Christophe Masselon, Ljiljana Paša-Tolić, Nikola Tolić, Gordon A...
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Anal. Chem. 2001, 73, 3312-3322

High-Throughput Peptide Identification from Protein Digests Using Data-Dependent Multiplexed Tandem FTICR Mass Spectrometry Coupled with Capillary Liquid Chromatography Lingjun Li, Christophe D. Masselon, Gordon A. Anderson, Ljiljana Pasˇa-Tolic´, Sang-Won Lee, Yufeng Shen, Rui Zhao, Mary S. Lipton, Thomas P. Conrads, Nikola Tolic´, and Richard D. Smith*

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

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable “on-the-fly” data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome. Biological mass spectrometry has emerged as a powerful analytical tool for proteomics due to its high sensitivity, speed, and capability for analysis of highly complex mixtures.1-3 An array of mass spectrometric techniques has been developed and applied to protein identification.1-10 These developments, coupled with the availability of the rapidly expanding protein and genomic data(1) Yates, J. R., III. J. Mass Spectrom. 1998, 33, 1-19. (2) Pandey, A.; Mann, M. Nature 2000, 405, 837-846. (3) Smith, R. D. Nat. Biotechnol. 2000, 18, 1041-1042. (4) Mann, M.; Hojrup, P.; Roepstorff, P. Biol. Mass Spectrom. 1993, 22, 338345.

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bases, have significantly speeded functional genomic studies at the protein level. Many of these approaches rely on digestion of an isolated protein or a small subset of proteins (e.g., obtained using 2D-PAGE) into peptides by a sequence-specific enzyme (e.g., trypsin), followed by either “peptide mass fingerprinting” using matrix-assistedlaserdesorption/ionization(MALDI)massspectrometry4-6 or tandem electrospray ionization (ESI) mass spectrometry.6-10 While the first approach is simple and high throughput, it often suffers from insufficient confidence for protein identification when multiple proteins are present or when extensive posttranslational modifications of the proteins of interest and/or sequence errors in the database occur. In contrast, the second strategy, incorporating gas-phase fragmentation in a mass spectrometer and subsequent database searching, provides more reliable protein identification. Since only a single (or at most a few) tryptic peptides from any given protein is required, this strategy enables identification of multiple proteins in mixtures and is highly tolerant of posttranslational modifications or database sequence errors.11,12 Therefore, intensive effort has been directed to peptide sequence analysis using tandem mass spectrometric techniques coupled with liquid chromatographic separations.13-18 Among others, triplestage quadrupole (TSQ) MS10,19 combined with very low flow rate (5) James, P.; Quadroni, M.; Carafoli, E.; Gonnet, G. Biochem. Biophys. Res. Commun. 1993, 195, 58-64. (6) Shevchenko, A.; Jensen, O. N.; Podtelejnikov, A. V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Shevchenko, A.; Boucherie, H.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445. (7) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233-6237. (8) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399. (9) Yates, J. R., III; Eng, J. K.; McCormack, A. L.; Schieltz, D. Anal. Chem. 1995, 67, 1426-1436. (10) Figeys, D.; Ducret, A.; Yates, J. R., III; Aebersold, R. Nat. Biotechnol. 1996, 14, 1579-1583. (11) Qin, J.; Feny, D., II; Zhao, Y.; Hall, W. W.; Chao, D. M.; Wilson, C. J.; Young, R. A.; Chait, B. T. Anal. Chem. 1997, 69, 3995-4001. (12) Yates, J. R., III; McCormack, A. L.; Eng, J. K. Anal. Chem. 1996, 68, 534A540A. (13) McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R., III. Anal. Chem. 1997, 69, 767-776. (14) Ducret, A.; van Oostveen, I.; Eng, J. K.; Yates, J. R., III; Aebersold, R. Protein Sci. 1998, 7, 706-719. (15) Davis, M. T.; Stahl, D. C.; Hefta, S. A.; Lee, T. D. Anal. Chem. 1995, 67, 4549-4556. (16) Emmett, M. R.; White, F. M.; Hendrickson, C. L.; Shi, S. D. H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998, 9, 333-340. 10.1021/ac010192w CCC: $20.00

© 2001 American Chemical Society Published on Web 06/14/2001

electrospray ionization (i.e., nanospray)20 has demonstrated attractive MS/MS capabilities. For example, MS/MS peptide analysis of low-femtomole 13 and subfemtomole protein identification using capillary zone electrophoresis 21 has been reported. Furthermore, Hunt and co-workers demonstrated MS/MS characterization of 10-20 fmol of immunologically relevant peptides from complex biological mixtures using capillary LC with a TSQ instrument.22,23 Quadrupole ion trap (i.e., QIT) mass spectrometers have gained popularity for MS/MS in recent years’ peptide analysis17,24-26 due to their ease of operation and high MS/MS sensitivity. For example, attomole-range sequencing of peptides has been demonstrated using ion trap mass spectrometers.27,28 While it is well established that low-resolution MS/MS measurements employing either TSQ or QIT instrumentation enable peptide identification, these approaches suffer from relatively poor mass accuracy (generally no better than 100 ppm with on-line separations) that can make identifications less confident. As noted above, quadrupole instruments suffer from relatively low sensitivity that results from their m/z scanning nature and ion traps suffer from a small dynamic range that arises from their limited charge capacity.29 More recently, hybrid quadrupole time-of-flight instruments have attracted considerable attention due to their improved mass measurement accuracy (MMA, ∼5-50 ppm depending upon peak intensity) and higher MS/MS sensitivity than TSQ instruments.30,31 An attractive alternative for proteomic studies is Fourier transform ion cyclotron resonance (FTICR) mass spectrometry which simultaneously provides high MMA, high resolving power, high sensitivity, and MS/MS capabilities.32-37 Numerous reports (17) Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C.; Yates, J. R., III. Anal. Chem. 2000, 72, 757-763. (18) Martin, S. E.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. Anal. Chem. 2000, 72, 4266-4274. (19) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858. (20) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (21) Figeys, D.; van Oostveen, I.; Ducret, A.; Aebersold, R. Anal. Chem. 1996, 68, 1822-1828. (22) Wang, W.; Meadows, L. R.; den Haan, J. M.; Sherman, N. E.; Chen, Y.; Blokland, E.; Shabanowitz, J.; Agulnik, A. I.; Hendrickson, R. C.; Bishop, C. E. Science 1995, 269, 1588-1590. (23) Cox, A. L.; Skipper, J.; Chen, Y.; Henderson, R. A.; Darrow, T. L.; Shabanowitz, J.; Engelhard, V. H.; Hunt, D. F.; Slingluff, C. L., Jr. Science 1994, 264, 716-719. (24) Marina, A.; Garcia, M. A.; Albar, J. P.; Yague, J.; Lopez de Castro, J. A.; Vazquez, J. J. Mass Spectrom. 1999, 34, 17-27. (25) Yates, J. R., III; Morgan, S. F.; Gatlin, C. L.; Griffin, P. R.; Eng, J. K. Anal. Chem. 1998, 70, 3557-3565. (26) Qin, J.; Herring, C. J.; Zhang, X. Rapid Commun. Mass Spectrom. 1998, 12, 209-216. (27) Settlage, R. E.; Russo, P. S.; Shabanowitz, J.; Hunt, D. F. J. Microcolumn Sep. 1998, 10, 281-285. (28) Gucˇek, M.; Gaspari, M.; Walhagen, K.; Vreeken, R. J.; Verheij, E. R.; van der Greef, J. Rapid Commun. Mass Spectrom. 2000, 14, 1448-1454. (29) Li, G.-Z.; Guan, S.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998, 9, 473-481. (30) Morris, H. R.; Paxton, T.; Panico, M.; McDowell, R.; Dell, A. J. Protein Chem. 1997, 16, 469-479. (31) Kristensen, D. B.; Imamura, K.; Miyamoto, Y.; Yoshizato, K. Electrophoresis 2000, 21, 430-439. (32) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (33) Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9075-9078. (34) Hendrickson, C. L.; Emmett, M. R. Annu. Rev. Phys. Chem. 1999, 50, 517536. (35) Smith, R. D. Int. J. Mass Spectrom. 2000, 200, 509-544.

have described the utility of FTICR for accurate mass measurements of biopolymers38,39 as well as the identification of polypeptides and proteins via a variety of activation schemes.40-45 Furthermore, the dynamic range of FTICR analysis is more than 2 orders of magnitude greater than for ion traps and is further improved by on-line coupling with chromatographic separations.16,18,46,47 Due to the complexity of the proteomic samples (e.g., from whole proteome digestions, which can consist of >105 different polypeptides),2,3 multiple peptides generally coelute even for the highest resolution LC separations. On the other hand, conventional MS/MS analysis is sequential (i.e., can only address one peptide at a time), and the data acquisition rate typically fails to allow selection and fragmentation of all detected peptides in the time available for analysis (i.e., the elution time of a peak). Consequently, the dynamic range is effectively reduced since typically the low-abundance ions are not selected for MS/MS analysis even when “dynamic exclusion” methods are used to prevent repetitive selection for MS/MS of the same peak. To help alleviate the problem, various “peak-parking” schemes have been developed in which the chromatographic peak elution time is extended to allow additional MS/MS experiments to be conducted.15,48,49 For example, Martin et al. recently described a variable-flow HPLC apparatus for on-line tandem mass spectrometric analysis of tryptic peptides.18 While such an approach alleviates the problem to some extent, comprehensive MS/MS analysis remains impractical for complex proteome digest samples. The reduction of LC flow rates not only significantly increases the overall separation time but may also decrease sensitivity for MS detection (particularly for the use of very small inner diameter capillaries where the electrospray ionization efficiency is already close to its maximum50) and the resolution of the separations and is less useful for high-performance separations that are based upon the use of very high pressures. (36) Pasˇa-Tolic´, L.; Jensen, P. K.; Anderson, G. A.; Lipton, M. S.; Peden, K. K.; Martinovic´, S.; Tolic´, N.; Bruce, J. E.; Smith, R. D. J. Am. Chem. Soc. 1999, 121, 7949-7950. (37) Jensen, P. K.; Pasˇa-Tolic´, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.; Bruce, J. E.; Smith, R. D. Anal. Chem. 1999, 71, 2076-2084. (38) Kelleher, N. L.; Senko, M. W.; Siegel, M. M.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1997, 8, 380-383. (39) Bruce, J. E.; Anderson, G. A.; Wen, J.; Harkewicz, R.; Smith, R. D. Anal. Chem. 1999, 71, 2595-2599. (40) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (41) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012808. (42) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (43) McCormack, A. L.; Jones, J. L.; Wysocki, V. H. J. Am. Soc. Mass Spectrom. 1992, 3, 859-862. (44) Jockusch, R. A.; Schnier, P. D.; Price, W. D.; Strittmatter, E. F.; Demirev, P. A.; Williams, E. R. Anal. Chem. 1997, 69, 1119-1126. (45) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573. (46) Li, W.; Hendrickson, C. L.; Emmett, M. R.; Marshall, A. G. Anal. Chem. 1999, 71, 4397-4402. (47) Shen, Y.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K.; Pasˇa-Tolic´, L.; Veenstra, T. D.; Lipton, M. S.; Udesth, H. R.; Smith, R. D. Anal. Chem. 2001, 73, 1766-1775. (48) Davis, M. T.; Lee, T. D. J. Am. Soc. Mass Spectrom. 1998, 9, 194-201. (49) Goodlett, D. R.; Wahl, J. H.; Udseth, H. R.; Smith, R. D. J. Microcolumn Sep. 1993, 5, 57-62. (50) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem., 1993, 65, 574A-584A.

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In this paper, we describe an alternative approach that utilizes the multiplexing capability and high MMA of FTICR for the simultaneous MS/MS analysis of multiple peptides during online capillary LC separations. A unique attribute of FTICR is the ability to select and simultaneously dissociate multiple precursor peptides. Previous “multiplexing” MS/MS approaches using FTICR were based upon comprehensive 2D studies51 and Hadamard transform methods.52 While repeating MS/MS experiments with different subsets of parent ions allow the assignment of all fragments to the corresponding parent species, these methods require a large amount of sample and are too slow for the use with on-line separations. Furthermore, a more significant problem associated with these 2D approaches is that it has proved impractical to generate comparable degrees of dissociation for different m/z ions from a mixture. More recently, our laboratory reported a multiplexed-MS/MS strategy in which multiple peptides are dissociated in the ICR cell and the fragments measured with a high MMA so that amino acid sequence information for multiple peptides is obtained simultaneously, providing both enhanced sensitivity and a gain in throughput.53 Herein we demonstrate on-the-fly data-dependent selection of a subset of polypeptides from each FTICR spectral acquisition during the LC separation and their simultaneous dissociation and measurement in the subsequent (i.e., MS/MS) acquisition. We have initially evaluated both the sustained off-resonance irradiation collisionally induced dissociation (SORI-CID)40,41 and infrared multiphoton dissociation (IRMPD)42 methods with on-line capillary LC multiplexed-MS/MS. For IRMPD experiments, all selected species were retained in the ICR trap and dissociated using infrared radiation from a CO2 laser. In the case of SORI-CID, multiple activation frequencies were applied simultaneously, resulting in fragmentation of different peptides using a single waveform. The utility of this approach has been demonstrated using bovine serum albumin (BSA) tryptic digest separated by capillary reversed-phase (RP) LC, where multiple peptides were readily identified in a single tandem mass spectrum. Additionally, initial results for a more complex proteome sample, a global tryptic digest of Deinococcus radiodurans proteins, using the multiplexed SORI-CID approach are presented to demonstrate the utility of this approach for high-throughput proteomic analysis. EXPERIMENTAL SECTION Sample Preparation. BSA Digest. BSA was obtained from Sigma (St. Louis, MO) and used without further purification. A 1-mg sample of BSA was dissolved in 1 mL of PBS (pH 7.5) and 5 mM DTT. To ensure denaturation, urea was added to a final concentration of 8 M, and the solution was heated to 100 °C for 5 min. After cooling, the solution was diluted 10-fold with 50 mM NH4HCO3 (pH 7.8), 1 mM CaCl2 to ensure the urea concentration was below 1 M. Trypsin was dissolved in PBS and added in a protease-to-protein ratio of 1:25. The mixture was then incubated at 37 °C overnight. After incubation, the digest solution was dialyzed in a 500-Da MW cutoff dispodialyzer tube (Fisher) against (51) Ross, C. W.; Guan, S. H.; Grosshans, P. B.; Ricca, T. L.; Marshall, A. G. J. Am. Chem. Soc. 1993, 115, 7854-7861. (52) Williams, E. R.; Loh, S. Y.; McLafferty, F. W.; Cody, R. B. Anal. Chem. 1990, 62, 698-703. (53) Masselon, C.; Anderson, G. A.; Harkewicz, R.; Bruce, J. E.; Pasˇa-Tolic´, L.; Smith, R. D. Anal. Chem. 2000, 72, 1918-1924.

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50 mM Tris (pH 7.0) overnight to remove excess urea. The peptide mixture was assayed by the Lowry method (BCA kit, Pierce, Rockford, IL) to determine peptide concentration. Preparation of Cellular Tryptic Digest from D. radiodurans. D. radiodurans cells were cultured in TGY medium to an approximate OD600 of 1.2 and harvested by centrifugation at 10000g at 4 °C. Prior to lysis, cells were resuspended and washed three times with 100 mM ammonium bicarbonate and 5 mM EDTA (pH 8.4). The cells were lysed by beating with 0.1-mm acid zirconium beads using three 1-min cycles at 5000 rpm. Between each cycle of bead beating, the samples were incubated on ice for 5 min. The supernatant containing soluble cytosolic proteins was recovered after centrifugation at 15000g for 15 min to remove cell debris. Proteins were denatured and reduced by addition of guanidine hydrochloride (6 M) and DTT (1 mM), respectively, followed by boiling for 5 min. Prior to trypsin digestion, the protein sample was desalted using a 5000 molecular weight cutoff “D-salt” gravity column (Pierce, Rockford, IL) equilibrated in 100 mM ammonium bicarbonate (pH 8.4). Proteins were enzymatically digested at an enzyme/protein ratio of 1:50 (w/w) using sequencing grade modified trypsin (Promega, Madison, WI) at 37 °C for 16 h. Capillary RPLC Coupled with ESI-FTICR MS. Various HPLC grade solvents were purchased from Aldrich (Milwaukee, WI). Fused-silica capillary columns (30-60 cm, 150 µm i.d. × 360 µm o.d., Polymicro Technologies, Phoenix, AZ) packed with 5-µm C18 particles were manufactured in-house as described previously.47 Capillary RPLC was performed using either a Shimadzu LC-10AD system or an ISCO LC system (model 100DM, ISCO, Lincoln, NE). A microflow splitter (LC packings, San Francisco, CA) produced a gradient at a flow rate of ∼2 µL/min. The mobile phases for gradient elution were (A) acetic acid/TFA/water (0.2: 0.05:100 v/v) and (B) TFA/acetonitrile/water (0.1:90:10, v/v). With the Shimadzu system, after a sample volume of 10 µL was injected onto the RPLC column, a linear gradient was used from mobile phase A to 80% B in 60 min and then to 100% B in another 15 min. The column reequilibration was carried out using 100% A for more than 1 h. In the case of the ISCO system, the mobile phases, delivered at 5000 psi using two ISCO pumps, were mixed in a stainless steel mixer (∼2.8 mL) with a magnetic stirrer before entering the separation capillary. Fused-silica capillary flow restrictors (30-µm i.d. with various lengths) were used to manipulate the gradient speed. Eluent from the capillary RPLC column was electrosprayed into the FTICR mass spectrometer by a spray emitter that was connected to the column via a stainless steel union. The 7-T FTICR mass spectrometer utilized in this work has been described in detail previously.54 In brief, the instrument comprises a custom-built ESI source consisting of a heated “desolvation” inlet capillary interfaced with an electrodynamic ion funnel55,56 to achieve high ion transmission efficiency from the atmospheric region to a short rf quadrupole for collisional focusing. Two subsequent sets of rf-only quadrupoles (∼1.2 MHz, (54) Winger, B. E.; Hofstadler, S. A.; Bruce, J. E.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 566-577. (55) Kim, T.; Tolmachev, A. V.; Harkewicz, R.; Prior, D. C.; Anderson, G. A.; Udseth, H. R.; Smith, R. D.; Bailey, T. H.; Rakov, S.; Futrell, J. H. Anal. Chem. 2000, 72, 2247-2256. (56) Belov, M. E.; Gorshkov, M. V.; Udseth, H. R.; Anderson, G. A.; Tolmachev, A. V.; Prior, D. C.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2000, 11, 19-23.

Figure 1. (A) A diagram of the major components of the data-dependent control system used for LC-FTICR multiplexed-MS/MS experiments (see Experimental Section). (B) Electronic connections used for data-dependent control experiments. Relay 1 is used to send normal excitation or SWIFT/SORI signal to the excitation amplifier (See Experimental Section for details). The data acquired from the ICR cell preamplifier are transferred in parallel to the Odyssey data station for storage and to the ancillary control PC for generating and downloading the SWIFT and SORI waveforms to be used in the following MS/MS acquisition.

∼300 Vpp) located in higher vacuum regions guide ions to the ICR cell. Mass spectra were acquired using standard experimental sequences (i.e., ion injection and accumulation, pump-down, and excitation/detection). Background pressure in the ICR cell was maintained at ∼10-9 Torr by a custom cryopumping assembly that provides pumping speeds of ∼105 L/s, which allowed rapid transition between in-trap ion accumulation (i.e., 10-5 Torr) and high-performance ion excitation/detection (i.e., 10-9 Torr) events. A piezoelectric pulse valve (Lasertechniques, Albuquerque, NM) was used to inject N2 gas (to ∼10-5 Torr) for accumulated trapping of ions and for ion activation in CID experiments. An Odyssey data station (Finnigan, Madison, WI), running software version 4.0, controls ion injection, excitation (i.e., broadband chirp excitation over a 100-kHz bandwidth with a 35 Hz/µs sweep rate), and detection (256K data points at 270 kHz), followed by data storage. Data-Dependent FTICR Multiplexed-MS/MS. Data-dependent control was accomplished using the Odyssey data station coupled with an ancillary PC operating under the ICR-2LS software developed in our laboratory.57 The Odyssey data station was used to provide the experiment scripts (i.e., to control and generate all of the timing signals, potentials, and transistor-transistor-logic (TTL) trigger signals; see Figure 1). The combined capillary LCFTICR experiment consisted of continuous repetition of two acquisitions: MS followed by MS/MS. During the MS acquisition, the PC was triggered to acquire raw data in parallel with the data station via a National Instruments 6070E Analog I/O card. The ICR-2LS program converted the time domain raw data to an m/z spectrum during the experiment and rapidly generated an appropriate ion isolation waveform (e.g., stored-waveform inverse Fourier transform, SWIFT58,59) and ion excitation waveform (e.g., SORI40) that were then downloaded to a National Instruments

DAQ 5411 arbitrary waveform generator. Prior to the subsequent MS/MS acquisition, this arbitrary waveform generator was triggered by a TTL signal from the data station to isolate the selected precursor ion(s) and (optionally) activate them during the CID step. As shown in Figure 1B, relay 1 controlled the signal that was sent to the excitation amplifier. In its default position, the data station excitation signal was sent to the excitation amplifier and then to the detection plates of the cell. When this relay was energized, the SWIFT isolation and SORI excitation (used for CID experiments only) signals from the arbitrary waveform generator National Instruments DAQ5411 were sent through the excitation amplifier to the ICR cell excitation plates. The analog I/O card acquired the time domain signal when an external trigger was detected. After the acquisition, ICR-2LS processed the data on the basis of a series of user predefined processing steps. A log file, containing a list of selected parent ion masses and the SORI excitation parameters used, was created for each LC-FTICR MS/MS analysis to assist future data analysis. Multiple precursor ions can be selected for subsequent dissociation based on different user-defined criteria (e.g., relative abundance). The dynamic exclusion option (a user-defined signal intensity exclusion threshold) was used to prevent reacquisition of tandem mass spectra of ions for which an MS/MS spectrum has already been acquired during a given chromatographic time period (e.g., 1 min). This functionality allows for maximization of the number of peptides that can be identified in a single LC run by reducing the redundant fragmentation, thus further enhancing (57) ICR2LS; Anderson, G. A., Bruce, J. E., Eds.; Pacific Northwest National Laboratory: Richland, WA, 1995. (58) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (59) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 5-37.

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Figure 2. Total ion chromatogram reconstructed from the FTICR spectra obtained during the capillary RPLC separation of BSA tryptic digest and examples of multiplexed SORI-CID spectra (four parent species selected after each MS acquisition based on relative ion abundances) obtained at the indicated points during the LC separation.

the throughput for multiplexed-MS/MS analysis. For multiplexed SORI-CID experiments, in addition to the generation of SWIFT waveform for isolation of multiple parent ions, multiple frequency SORI waveforms were generated as a superposition of the individual SORI waveforms.60 The average collisional energy was controlled by varying either the rf excitation voltages (∼3-5 Vpp) or the frequency offset between cyclotron and excitation frequency (typically 1000 Hz off-resonance) with irradiation duration of 500 ms. For the on-line LC multiplexed IRMPD experiments, a CO2 laser (model 48-2W-25W, Synrad, Mukilteo, WA) operated in the pulsed mode was used for dissociation of isolated peptides by gating the laser controller with a TTL trigger delivered by the FTICR Odyssey data station. The laser controller voltage was set at 4.25 V, corresponding to ∼15 W, with irradiation time of 300 ms. The beam passed through a ZnSe beam splitter and was directed on the magnetic field axis of the FTICR instrument by means of silver-coated silicon mirrors (TRS1.0-PS-0339, Laser Power Optics, San Diego, CA). The beam entered the instrument through a BaF2 window (Bichron Harshaw, Solon, OH) mounted on a miniflange. (60) Hofstadler, S. A.; Wahl, J. H.; Bakhtiar, R.; Anderson, G. A.; Bruce, J. E.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1994, 5, 894-899.

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Data Analysis and Database Searching. Analysis of the large data sets arising from capillary RPLC FTICR experiments was performed in an automated fashion using ICR-2LS.57 Time domain signals were apodized (Hanning) and zero-filled twice before fast Fourier transform to produce mass spectra. Isotopic distributions and charge states in the mass spectra were deconvolved using the THRASH algorithm developed by Horn et al.61 The large ion populations lead to significant space charge effects and reduced MMA compared to that typically obtained with smaller ion populations using the present 7-T FTICR. The recently described DeCAL method of calibration was used to reduce the effects of space charge-induced shifts and improve MMA without the requirement for internal calibrants.62 The m/z values of the parent ions and of the fragments were then included as two separate lists into a “Find Protein” module of the ICR-2LS software package, which performed the database search. The databases used included the BSA sequence, the SwissProt database (downloaded from http://expasy.cbr.nrc.ca/sprot/ and converted to a Microsoft Access molecule database that contains identification number, (61) Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 2000, 11, 320-332. (62) Bruce, J. E.; Anderson, G. A.; Brands, M. D.; Pasˇa-Tolic´, L.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2000, 11, 416-421.

Figure 3. FTICR MS and multiplexed-MS/MS (SORI-CID) spectra obtained for a BSA tryptic digest during a capillary RPLC separation. (A) MS spectrum showing the four selected precursor ions (since dynamic exclusion was activated, selected ions do not correspond to the highest abundant ones), with the masses labeled being the calculated neutral masses of the selected species. (B) Multiple frequency SORI-CID spectrum showing the product ions resulting from simultaneous dissociation of the four selected m/z species (corresponding to three neutral masses). The symbols indicate the fragment attribution for the three identified peptides.

protein sequence, molecular masses, etc.) of over 77 000 proteins, and a D. radiodurans predicted protein database derived from genome sequence data (downloaded from ftp://ftp.tigr.org/pub/ data/d_radiodurans/). The peptide search algorithm was described previously.53 Briefly, a list of all possible tryptic peptides was generated from the appropriate database. The measured mass for each parent species (assuming a very conservative 25 ppm MMA) was then searched against all masses for species on this list of possible tryptic digestion products, resulting in a set of “candidates” for each parent species. The subsequent search was performed using only a set of predicted fragment ion species originating from the possible “candidate” peptides. The list of possible fragment species for the candidates included all of the b and y fragments as well as product ions corresponding to the loss of water or ammonia from these same b and y fragments. For all candidates, possible fragment masses were computed and compared to the list of masses from the LC multiplexed-MS/MS data (again assuming 25 ppm MMA). RESULTS AND DISCUSSION In contrast to conventional MS/MS approaches wherein peptides are sequentially selected and dissociated one at a time,

FTICR offers the capability to simultaneously select and dissociate multiple precursor ions. We previously reported the use of a multiplexed tandem MS approach to simultaneously identify (i.e., from one mass spectrum) a mixture of seven polypeptides using IRMPD.53 The applicability of this approach to complex mixture analysis was initially limited by the need for operator intervention during data acquisition (i.e., to perform ion isolation and activation). Recently, a few examples of FTICR data-dependent acquisition schemes were presented.37,63-65 The current study exploits new automated instrument control software developed at our laboratory for tandem FTICR experiments to enable the use of a multiplexed-MS/MS approach for on-line separations. A complex polypeptide mixture resulting from the tryptic digestion of BSA was used to evaluate the applicability of the data-dependent multiplexed-MS/MS approach. Initial results from the LC-FTICR (63) Guan, S.; Marshall, A. G. Anal. Chem. 1997, 69, 1-4. (64) Berg, C.; Speir, P.; Kruppa, G.; Afzaal, S.; Laukien, F. Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, 1998; p 515. (65) Senko, M. W.; White, F. M.; Syka, J. E. P.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, 1999; p 2500.

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multiplexed-MS/MS analysis of a complex bacterial proteomic sample are also presented to demonstrate the significantly enhanced throughput for protein identification using this methodology. As IRMPD and SORI-CID are presently the most frequently used dissociation techniques in conjunction with FTICR, and both approaches sample the lowest energy dissociation pathways, we evaluated both activation methods with the multiplexed-MS/MS experiments in the current study. The multiplexed SORI-CID experiments take advantage of the superposition principle, wherein multiple SORI activation frequencies can be applied simultaneously and will act independently of each other.41,60 Consequently, the application of a single SORI waveform composed of multiple irradiation frequencies yields fragmentation of a selected subset of species at different m/z values. In this study, we have applied multiple frequency SORI waveforms to dissociate chromatographic effluents as a function of recorded MS data during an LC-FTICR experiment. Figure 2 shows the total ion chromatogram reconstructed from the FTICR multiplexed-MS/MS spectra obtained during capillary RPLC separation of a BSA tryptic digest and examples of multiplexed SORI-CID spectra (four parent ions were simultaneously selected and dissociated based on the preceding MS acquisition). From spectra obtained for several representative chromatographic peaks, it is readily seen that extensive fragmentation occurred and a relatively wide dynamic range realized, as illustrated by the high-quality dissociation spectra obtained for both high- and low-abundance species. Two separate lists of monoisotopic masses resulting from the MS and MS/MS acquisitions were searched against a database containing all possible BSA tryptic fragments. The list of possible fragment species included b and y fragments and product ions corresponding to the loss of water or ammonia from these same b and y fragments for each of the possible polypeptides. Figure 3 shows an example of a multiplexed SORI-CID spectrum for a BSA tryptic digest obtained from a single acquisition event during an LC separation. As shown in Figure 3A, four different peaks were selected and dissociated simultaneously (note that two of the peaks selected correspond to different charge states of the same species). Multiplexed SORICID resulted in an extensive array of sequence-specific product ions, providing the basis for confident peptide identification. The three selected peptides were identified as LKPDPNTLCDEFKADEK, RPCFSALTPDETYVPK, and LVNELTEFAK from the BSA sequence (Figure 3B). Compared to SORI-CID, IRMPD has the advantage for on-line coupling with LC separation of obviating the need for introduction of a collision gas (which typically requires lengthening the experimental sequence by several additional seconds to evacuate the ICR cell to the base pressure) and thus offers an increased speed for MS/MS measurements. In addition, IRMPD also provides on-axis fragmentation and minimal mass discrimination.42,46 Figure 4A shows representative MS and corresponding IRMPD MS/MS spectra obtained during the capillary RPLC separation of a BSA tryptic digest. In this example, the three most abundant precursor ions were simultaneously selected using a SWIFT waveform (note that two of the selected m/z values correspond to the same parent mass; however, the exclusion of “double selections” can easily be incorporated into data-dependent peak selection software). As shown in the multiplexed-MS/MS 3318 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

Figure 4. Examples of FTICR MS and multiplexed-MS/MS (IRMPD showing in A and SORI-CID showing in B) spectra obtained from BSA tryptic digest during capillary RPLC separation. The three most abundant ions from the MS acquisition were selected using SWIFT, with the masses shown in parentheses being the calculated neutral masses for the detected species. Prior to the subsequent MS/MS acquisition, three selected ion species (two neutral masses) were dissociated using IRMPD (A) or SORI-CID (B); resulting fragments were attributed using BSA database search. Comparison of the fragmentation patterns of the peptide SLHTLFGDELCK obtained using IRMPD and SORI-CID is discussed in detail in the Results and Discussion section. The inset (B) shows the expanded view of m/z 950-1050.

spectrum obtained using IRMPD, most of the peaks were assigned to the sequence-specific fragment ions resulting from two BSA tryptic peptides: DAIPENLPPLTADFAEDK and SLHTLFGDELCK. While lower intensity dissociation products may be generated by IRMPD (e.g., the formation of internal fragments), no attempts were made to assign these fragments in the multiplexed-MS/MS spectrum. It is also interesting to note that a series of consecutive “y” ions was observed for both tryptic peptides. These give “sequence tags” that provide additional confirmation of the peptide identification (and often unambiguous protein identification). Table 1 lists the peptides identified from the LC-FTICR multiplexed-MS/MS data using the BSA sequence. The mass accuracy for both parent mass and fragment mass was assumed to be better than 25 ppm, and at least two MS/MS fragments were

Table 1. Identified Peptides for Tryptic Digestion of Bovine Serum Albumin Using Multiplexed MS/MS precursor mass (Da)

IRMPD

calc

exp

MMA (ppm)

788.464 817.418 840.453 885.408 921.481 926.486 973.450 976.444 1001.576 1013.612 1014.480 1049.485 1141.707 1162.623 1176.552 1192.595 1248.614 1282.703 1304.709 1361.665 1385.613 1398.685 1417.731 1438.804 1478.788 1496.624 1518.739 1566.735 1638.930 1666.806 1822.892 1849.892 1887.919 1887.988 1889.796 1954.952 1961.940 2044.021 2198.093 2434.235 2483.139 2866.177 3396.622 4129.961

788.462 817.413 840.447 885.399 921.493 926.482 973.445 976.445 1001.568 1013.608 1014.461 1049.477 1141.701 1162.615 1176.545 1192.577 1248.608 1282.695 1304.712 1361.667 1385.624 1398.682 1417.717 1438.797 1478.771 1496.622 1518.745 1566.727 1638.927 1666.801 1822.908 1849.890 1887.904 1887.970 1889.785 1954.945 1961.928 2044.049 2198.108 2434.248 2483.150 2866.159 3396.628 4129.985

2.5 6.1 7.1 10.2 -13.0 4.3 5.1 -1.0 8.0 3.9 18.7 7.6 5.2 6.9 5.9 15.1 4.8 6.2 -2.3 -1.5 -7.9 2.1 9.9 4.9 11.5 1.3 -3.9 5.1 1.8 3.0 -8.8 1.1 7.9 9.5 5.8 3.6 6.1 -13.7 -6.8 -5.3 -4.4 6.3 -1.8 -5.8

av

assigned sequence LVTDLTK ATEEQLK LCVLHEK DDSPDLPK AEFVEVTK YLYEIAR DLGEEHFK NECFLSHK LVVSTQTALA QTALVELLK SHCIAEVEK EACFAVEGPK KQTALVELLK LVNELTEFAK ECCDKPLLEK DTHKSEIAHR FKDLGEEHFK HPEYAVSVLLR HLVDEPQNLIK SLHTLFGDELCK YICDNQDTISSK TVMENFVAFVDK LKECCDKPLLEK RHPEYAVSVLLR LGEYGFQNALIVR DDPHACYSTVFDK LKPDPNTLCDEFK DAFLGSFLYEYSR KVPQVSTPTLVEVSR MPCTEDYLSLILNR RPCFSALTPDETYVPK LFTFHADICTLPDTEK HPYFYAPELLYYANK SLHTLFGDELCKVASLR VASLRETYGDMADCCEK DAIPENLPPLTADFAEDK LKPDPNTLCDEFKADEK RHPYFYAPELLYYANK ATEEQLKTVMENFVAFVDK GLVLIAFSQYLQQCPFDEHVK QEPERNECFLSHKDDSPDLPK EYEATLEECCAKDDPHACYSTVFDK SHCIAEVEKDAIPENLPPLTADFAEDKDVCK SHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAK

2.7

required for identification (based on simulation results). A total of 44 tryptic peptides were identified using multiplexed IRMPD and SORI-CID, with 18 peptides identified with both dissociation approaches. While the possibility of the differences in sample preparation (e.g., the completeness of tryptic digest may slightly vary) cannot be completely excluded, it is interesting to note that some of the peptides are uniquely identified using only one method. For example, smaller tryptic peptides were identified using IRMPD, whereas SORI-CID identified several additional larger peptides (i.e., multiple missed cleavages). One can speculate that these differences in peptide identifications may be due to the fact that greater losses are to be expected for smaller ions formed off-axis by CID, while the continuous “heating” of the product ions formed on-axis during IRMPD likely leads to secondary dissociation, accounting for its decreased effectiveness for producing larger peptides. Furthermore, we note that both IRMPD and SORI-CID provided roughly comparable MMA using external calibration with a 7-T instrument, with SORI-CID being slightly

SORI-CID

no.of frag

MMA (ppm)

no.of frag

MMA (ppm)

3

3.3

2 3 2

10.5 15 7

2 2 2

13.4 6.6 23.6

3 4

22.2 4

5 2 2 4 4 2 4 4 2

5.5 1.4 16.9 3 4.5 12.2 4.1 7.3 5.4

6

6

4 7

20.6 2.4

8 3 7 8 4 10

9.7 1.3 5.7 8.1 2.4 12.2

4 10

7.3 7.2

7

7.7

2 2 5 10 4 9 3 3

13.1 7.2 14.5 4.1 2.8 18.9 11 9.2

10 14 6 14 4 14 13 2

8.1 7.3 8.1 15 10.7 3.7 4.7 6

18 17 12 9 9

13.3 5.6 9.9 10.8 14.9

2 3

10.2 4.4

8 13 2 2 2 3 9 6

2.3 8.7 8.6 7.6 19.4 16.4 17.1 20.8

13

4.7

4.5

7.9

7.2

10.5

poorer (average error of ∼10 ppm compared to 7.9 ppm in the case of IRMPD) likely due to the off-axis product ion formation. Nonetheless, SORI-CID on average provided more useful MS/ MS fragments for peptide identification, consistent with previous observations that SORI-CID provides more efficient fragmentation.41,42 To provide a closer comparison of the two dissociation techniques, Figure 4B shows a multiplexed SORI-CID spectrum of the BSA tryptic digest where two peptides are identified in this single acquisition. Comparison of the dissociation pattern of peptide SLHTLFGDELCK in parts A and B of Figure 4 reveals similar sequence-specific fragment ions, even though the relative peak abundances vary dramatically. For example, y6-y9, b10, and b11 fragments were observed in both spectra. However, it is interesting to note the absence of y4 and y5 ions, which are fairly intense in the IRMPD spectrum, from the SORI-CID spectrum, and the detection of a consecutive series of “b” ions (b5-b9), absent from the IRMPD spectrum, in the SORI spectrum. The y4 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

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Figure 5. (A) Two-dimensional display reconstructed from the FTICR spectra obtained during a capillary LC-FTICR analysis of a global tryptic digest from a D. radiodurans cell lysate. Over 13 000 peptide “spots” (i.e., isotopic distributions) were detected in a single FTICR MS part of the experiment. (B) A mass spectrum (indicated by dotted line in 2-D plot shown in A), with four most abundant ions selected for the subsequent MS/MS acquisition labeled. (C) Multiplexed SORI-CID spectrum of the four peptide species selected in (B) with fragment ions attributed to each individual peptide after searching against the D. radiodurans protein database. The four proteins uniquely identified from this spectrum are listed in the inset box with their open reading frame reference number (e.g., DR1577). The numbers listed in parentheses after each peptide indicated the number of fragment ions detected for each tryptic peptide. An expanded view of m/z 650-760 with sequence-specific fragment ions labeled is shown as inset.

and y5 ions absent in the SORI-CID experiment may be attributed to either the mass discrimination of this technique or their possible origin from sequential dissociation processes for IRMPD.41,42 The lack of observation of a series of “b” ions using IRMPD is consistent with previous ion stability studies in which it was found that most b-type ions are much less kinetically stable than the y-type ions, resulting in the faster decay of “b” ions under continuing IR irradiation.42,66 Thus, IRMPD and SORI-CID provide similar yet complementary fragmentation information, and both allow effective protein identification. Although further optimization and modifications would allow improved fragmentation for the tandem MS experiments, the aim of the multiplexed-MS/MS experiments is not to obtain complete sequence coverage for each peptide but, rather, to use the sequence-specific fragments in (66) Aaserud, D. J.; Little, D. P.; O’Connor, P. B.; McLafferty, F. W. Rapid Commun. Mass Spectrom. 1995, 9, 871-876.

3320 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

combination with the database searching strategy to identify peptides in a higher throughput manner. Previous simulations have shown that even a small number of MS/MS fragments (∼3) can often provide unambiguous identification of the parent species from an appropriate database, and the number of fragments needed decreases as MMA is increased.53 To further evaluate the utility of the LC-FTICR multiplexedMS/MS approach for more complex proteome-wide protein identification, we have used the mass measurements for both parent species and their fragments obtained in SORI-CID and IRMPD experiments to search against the SwissProt database (downloaded from http://expasy.cbr.nrc.ca/sprot/ and converted to a Microsoft Access molecule database of protein sequences, molecular masses, etc.). The SwissProt database contained 77 977 proteins. Using the database-searching algorithm described previously, and assuming 25 ppm MMA for both parents and frag-

ments, with only b and y fragments allowed and a minimum of 3 MS/MS fragments required, 28 peptides were returned as top hits identifying either BSA or other serum albumin proteins from different organisms. Among these identifications, 12 peptides were unique to BSA, allowing its unambiguous identification from this data set. Several peptides yielded identification of multiple proteins, all of which belonged to the highly homologous serum albumin protein family (e.g., MPCTEDYLSLILNR called out sheep serum albumin in addition to BSA). Moreover, three other proteins in the SwissProt protein database in addition to BSA contain the identified tryptic peptide KVPQVSTPTLVEVSR. One interesting case was the identification of three isobaric peptides LGEYGFQNALLVR, LGEYGFQNAILVR, and LGEYGFQNALIVR (Mr ) 1478.788). Multiplexed SORI-CID of this parent ion yielded total of 12 fragment ions, including a consecutive series of y ions (y3y10) and several b ions, with average MMA for the product ions being 4.0 ppm. However, these three peptides have almost identical sequence (only the isobaric amino acids Leu and Ile at positions 10 and 11 vary in the following order: LL vs IL vs LI); thus, their unambiguous identification was impractical with the low-energy dissociation techniques employed here. The peptide LGEYGFQNALLVR resulted in identification of Felca and Canivora serum albumins; the peptide LGEYGFQNAILVR called out mouse serum albumin, whereas the peptide sequence LGEYGFQNALIVR identified BSA and pig serum albumin. As an initial demonstration of the use of the multiplexed-MS/ MS approach for protein identification from complex proteome samples, Figure 5A shows a 2-D display for the capillary LCFTICR analysis of tryptic peptides from D. radiodurans whole cell lysate, where the peptide “spots” are based on their molecular mass and LC elution time. Over 13 000 peptide “spots” were observed in this single analysis. Figure 5B shows an example of a mass spectrum obtained in a single MS acquisition corresponding to the dotted line in the 2-D display, while Figure 5C shows the corresponding (i.e., immediately following) multiplexed SORICID spectrum with the dissociation products from the four most abundant parent ions selected from spectrum shown in Figure 5B. Table 2 lists fragment ion assignments from this MS/MS spectrum based upon a search of the D. radiodurans protein database. A significant number of sequence-specific fragments were attributed to each selected peptide. The extensive fragmentation allowed the identification of the four selected tryptic peptides, with each identifying a unique D. radiodurans protein. For example, the peptide with Mr ) 1628.85 was identified as LLDSGMAGDNVGVLLR from elongation factor TU (DR2050 and DR0309, which happen to be duplicated open reading frames), and the peptide with Mr ) 1696.94 was identified as a tryptic fragment from glyceraldehyde-3-phosphate dehydrogenase (DR1343). We have noted that peptides having significant differences in abundances (visualized by the spot size in 2-D display and the ion intensity in MS spectrum) could be readily fragmented to yield useful sequence information. The comprehensive analysis of these results will be the subject of a separate paper. We previously described the utility of accurate mass tags (AMTs) for proteome-wide protein identification.67 This approach is based on the ability to determine molecular masses with (67) Conrads, T. P.; Anderson, G. A.; Veenstra, T. D.; Pasˇa-Tolic´, L.; Smith, R. D. Anal. Chem., 2000, 72, 3349-3354.

Table 2. Fragments of the Four Tryptic Peptides from D. radiodurans Identified in a Single LC FTICR Multiplexed SORI-CID Spectrum (See Figure 5)a [M + H]+

assignment

m/z

measured

calculated

error (ppm)

peptide

fragment

401.288 405.200 500.356 554.357 557.380 559.289 604.301 617.305 644.844 656.452 667.445 670.329 688.337 691.893 701.392 727.352 733.342 742.414 743.487 745.365 770.506 796.499 797.934 806.445 840.476 860.388 885.535 925.522 925.522 942.555 946.552 955.475 959.512 996.556 996.556 1013.59 1073.514 1088.557 1113.503 1125.614 1144.646 1187.639 1201.651 1212.573 1229.613 1253.707 1253.707 1288.68 1303.747 1307.695 1324.728 1342.683 1380.768 1383.768 1400.766 1437.738 1455.760 1501.824

401.288 1212.573 500.356 554.357 557.380 599.289 604.301 617.305 1288.678 656.452 667.445 670.329 688.337 1381.770 1400.766 727.352 733.342 1483.820 743.487 745.365 770.506 796.499 1593.85 1611.881 1678.935 860.388 885.535 925.522 925.522 942.555 946.552 955.475 959.512 996.556 996.556 1013.59 1073.514 1088.557 1113.503 1125.614 1144.646 1187.639 1201.651 1212.573 1229.613 1253.707 1253.707 1288.68 1303.747 1307.695 1324.728 1342.683 1380.768 1383.768 1400.766 1437.738 1455.760 1501.824

401.288 1212.557 500.356 554.355 557.3775 559.286 604.294 617.297 1288.668 656.446 667.439 670.323 688.334 1381.757 1400.775 727.345 733.337 1483.812 743.478 745.355 770.489 796.482 1593.875 1611.852 1678.925 860.382 885.516 925.511 925.525 942.537 946.536 955.474 959.505 996.548 996.562 1013.574 1073.494 1088.547 1113.489 1125.604 1144.615 1187.616 1201.636 1212.557 1229.584 1253.701 1253.663 1288.668 1303.722 1307.675 1324.738 1342.668 1380.750 1383.748 1400.775 1437.741 1455.752 1501.822

-1.0 13.1 -0.4 3.1 4.5 5.0 11.6 12.9 7.7 9.1 9.0 8.3 4.4 9.2 -6.5 9.6 6.8 5.4 12.1 13.4 22.1 21.3 -15.6 18.2 6.0 6.5 21.5 11.9 -3.2 19.1 16.9 1.0 7.3 8.0 -6.0 15.8 18.6 9.2 13.0 8.9 27.1 19.4 12.5 13.2 23.6 4.8 35.1 9.3 19.2 15.3 -7.5 11.2 13.1 14.3 -6.4 -2.1 5.5 1.33

C C C D B/C C D C C C D C C D B C D B B C C D A C B C C C D C A B D C D C C D C D C D C C C A D C B A A C B B B C C B

y3 b13 - NH3 y4 b5 y5 b6 - H2O y5 b6 y13 y6 b6 b7 - H2O b7 M - H2O y14 b8 - H2O y6 y15 - H2O y7 b8 y7 b7 y15 M - H2O M - H2O b9 y8 y9 - NH3 b8 y9 y8 b10 y8 y10 - NH3 b9 y10 b11 y9 b12 - NH3 b10 y11 y10 y12 b13 - NH3 b13 y11 b11 y13 y13 b14 y12 b14 M - H2O y14 - NH3 y14 b15 - H2O b15 y15

a Peptide assignment corresponds to the open reading frame reference number listed in the inset box in Figure 5C.

sufficiently high MMA so that a significant fraction of these masses are unique among all of the possible peptides predicted for the system under study. In practice, we validate AMTs based upon MS/MS measurements and extend the utility of accurate mass measurements by also using the distinctive elution time for Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

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the peptide. We have shown elsewhere that the use of higher magnetic fields for FTICR facilitates routine FTICR measurement with 105 and provide close to 100% ion utilization efficiency.68,69 The sensitivity and dynamic range gains from the external accumulation of ions can be further extended by the application of data-dependent “active” FTICR dynamic range enhancement methods using external ion m/z selection. Toward this end, we have recently explored two different modes of ion preselection, using either rf/dc quadrupole filtering or rf-only dipolar excitation69 to achieve on-the-fly ion selection, followed by ion accumulation in a linear 2-D quadrupole trap external to the FTICR cell. The ability to selectively eject the most abundant species prior to external ion accumulation and the transfer of ions to the ICR cell should allow us to obtain broader proteome coverage by measuring low-abundance species that are undetectable using other methodologies. The effective gain in dynamic range can exceed 2 orders of magnitude.69 The coupling of this active dynamic range enhancement methodology with the multiplexed-MS/MS provides a basis for protein identification to be performed with much greater sensitivity and speed. This then largely obviates the need for MS/MS in subsequent studies where the validated AMT strategy can be combined with the use of stable-isotope labeling to enable quantitative and high-throughput proteome-wide measurements. CONCLUSIONS The utility of a data-dependent FTICR multiplexed-MS/MS approach in conjunction with on-line separations for high(68) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (69) Belov, M. E.; Nikolaev, E. N.; Anderson, G. A.; Auberry, K. J.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2001, 12, 38-48.

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Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

throughput peptide and protein identification has been demonstrated using a mixture of polypeptides resulting from digestion of bovine serum albumin, as well as the much more complex case of a global tryptic digest of a microbial proteome (from a D. radiodurans cell lysate). The data-dependent multiplexed-MS/MS approach, using two low-energy dissociation techniques, IRMPD and SORI-CID, has been successfully implemented to allow multiple peptides to be selected and dissociated simultaneously in real time during on-line capillary LC separations. While both approaches allow effective protein identification, SORI-CID provides more efficient fragmentation (in particular, more sequencespecific information) and is simpler to implement (no hardware modifications are required). Compared to the conventional MS/ MS approach, where individual peptides are selected and dissociated sequentially, the multiplexed-MS/MS approach allows multiple peptide species to be studied simultaneously, which not only decreases the required sample amount (i.e., increases sensitivity) but also enables peptide and protein identification from complex biological samples to be performed in a higher throughput manner. The high MMA and resolution provided by FTICR increase the confidence of protein identification. Furthermore, the multiplexing concept and strategy presented here should be readily adaptable to other FTICR dissociation schemes, but is more effective for the low-energy dissociation processes of the type studied here where fragmentation is limited, thus allowing more peptides to be simultaneously selected and their dissociation products observed with greater signal-to-noise ratios. Finally, we note that multiplexed-MS/MS of many more species than used in this initial demonstration is feasible and, together with a dynamic exclusion functionality and the dynamic range expansion methodology, should further increase the information and overall throughput for protein identification. We are currently extending this high-throughput multiplexed-MS/MS approach to more complex mammalian proteomes. ACKNOWLEDGMENT Portions of this work were supported by the National Cancer Institute under Grant CA86340 and the Office of Biological and Environmental Research, United States Department of Energy. Pacific Northwest National Laboratory is operated by the Battelle Memorial Institute for the U.S. Department of Energy through Contract DE-ACO6-76RLO 1830.

Received for review February 15, 2001. Accepted May 7, 2001. AC010192W