Identification of Bacteriophage MS2 Coat Protein from E. coli Lysates

reactions, provided the necessary sequence tag informa- tion (and molecular weight data) needed for protein identification via database searching. The...
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Anal. Chem. 2001, 73, 1277-1285

Identification of Bacteriophage MS2 Coat Protein from E. coli Lysates via Ion Trap Collisional Activation of Intact Protein Ions Benjamin J. Cargile,† Scott A. McLuckey,‡ and James L. Stephenson, Jr.*,§

Department of Biochemistry, University of Illinois UrbanasChampagne, Urbana, Illinois 61801, Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393, and Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365

Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E. coli lysates. Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion. Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server. These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein. These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap. The rapid analysis of complex protein mixtures represents one of the biggest challenges in all of biology. Complex protein mixture analysis is driven in no small part by the completion of genomic sequencing projects1,2 and the desire to understand the complex biological mechanisms associated with gene expression and protein regulation. Two basic strategies exist for the analysis of complex protein mixtures. The first involves the separation and identification of as many protein components as possible. This * Corresponding author: (phone) (423) 574-2848; (fax) (423) 576-8559; (e-mail) [email protected]. † University of Illinois UrbanasChampagne. ‡ Purdue University. § Oak Ridge National Laboratory. (1) Fleischmann, R.; Adams, M.; White, O.; Clayton, R.; Kirkness, E.; Kerlavage, A.; Bult, C.; Tomb, J.; Dougherty, B.; Merrick, J.; et al. Science 1995, 269, 496-512. (2) Bussey, H.; Storms, R. K.; Ahmed, A.; Albermann, K.; Allen, K.; Ansorage, W.; Araujo, R.; Aparicio, A.; Barrell, B.; Badcock, K.; et al. Science 1995, 269, 496-512. 10.1021/ac000725l CCC: $20.00 Published on Web 02/16/2001

© 2001 American Chemical Society

strategy lends itself readily to the field of proteomics, where it is desirable to obtain a comprehensive “snapshot” of the expressed proteins (ideally including quantitative data as well) in an organism at a given moment in time.3 The second approach involves the rapid identification of one or several protein component(s) in a complex mixture. For example, this approach is best utilized for the identification of microorganisms,4-14 identification of the specific stage of a disease state, or detection of a protein of interest from a well-defined biological system. Within the past decade, mass spectrometry has played an increasingly important role in identifying proteins from complex mixtures. In a recent report by Jensen et al., capillary isoelectric focusing (CIEF) was combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry in order to analyze 400-1000 putative proteins in the Escherichia coli proteome using a sample size of ∼300 ng.15 In addition, solid-phase microextraction/multistep elution/capillary electrophoresis/ tandem mass spectrometry was used to successfully identify 75 proteins from the yeast (Saccharomyces cerevisiae) ribosome.16 In the field of microorganism identification, matrix-assisted laser (3) Yates, J. R., III. J. Mass Spectrom. 1998, 33, 1-19. (4) Arnold, R. J.; Karty, J. A.; Ellington, A. D.; Reilly, J. P. Anal. Chem. 1999, 71, 1990-1996. (5) Welham, K. J.; Domin, M. A.; Scannell, D. E.; Cohen, E.; Ashton, D. S. Rapid Commun. Mass Spectrom. 1998, 12, 176-180. (6) Thomas, J. J.; Falk, B.; Fenselau, C.; Jackman, J.; Ezzell, J. Anal. Chem. 1998, 70, 863-867. (7) Wall, D. B.; Lubman, D. M.; Flynn, S. J. Anal. Chem. 1999, 71, 38943900. (8) Krishnamurthy, T.; Ross, P. L.; Rajamani, U. Rapid Commun. Mass Spectrom. 1996, 10, 883-888. (9) Dai, Y.; Li, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass Spectrom. 1999, 13, 73-78. (10) Haag, A. M.; Taylor, S. N.; Johnston, K. H.; Cole, R. B. J. Mass Spectrom. 1998, 33, 750-756. (11) Holland R. D.; Wilkes J. G.; Rafii F.; Sutherland J. B.; Persons C. C.; Voorhees K. J.; Lay J. O.; Rapid Commun. Mass Spectrom. 1996, 10, 1227-1232. (12) Claydon M. A.; Davey S. N.; Edwards-Jones V.; Gordon, D. B. Nature Biotechnol. 1996, 14, 1584-1586. (13) Demirev, P. A.; Ho, Y. P.; Ryzhov, V.; Fenselau, C. Anal. Chem. 1999, 71, 2732-2738. (14) Krishnamurthy, T.; Rajamani, U.; Ross, P. L.; Jabhour, R.; Nair, H.; Eng, J.; Yates, J.; Davis, M. T.; Stahl, D. C.; Lee, T. D. J. Toxicol.-Toxin Rev. 2000, 19, 95-117. (15) 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. (16) Tong, W.; Link, A.; Eng, J. K.; Yates, J. R. Anal. Chem. 1999, 71, 22702278.

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desorption/ionization (MALDI) coupled with time-of-flight (TOF) mass spectrometry has been used extensively to identify whole bacteria via protein fingerprinting.4,6,8,10,17 Karty et al. used the MALDI fingerprinting approach for development of a protein target analysis for the detection of the F plasmid (bacteriological sex factor) in various strains of E. coli.17 A recent targeted protein study employing bimolecular interaction analysis mass spectrometry18 was used to selectively isolate, detect, and characterize epitope-tagged peptides from whole-cell lysates. Masses obtained via MALDI-TOF of the epitope-tagged tryptic digest fragments were identified via a protein database search using the Genepept protein database.18 Complementary to the MALDI approach for microorganism identification, electrospray ionization in conjunction with “global” MS/MS in a quadrupole ion trap has been successfully used to differentiate the presence of two different microorganisms in a complex mixture.19 The combination of collisional activation of peptides (from proteolytic digest) and subsequent protein database searching has also evolved into a powerful tool for the identification of individual proteins from complex mixtures.20-23 When combined with online electrospray/mass spectrometry (ES/MS), separation procedures including those using microfabricated devices,24,25 microscale liquid chromatography mass spectrometry (LC/MS),26,27 two-dimensional LC/MS,28 and various capillary electrophoretic techniques coupled to MS15,29 make possible the analysis and identification of femtomole levels of protein from complex mixtures. However, the rate-limiting step in many of these highthroughput procedures is the proteolytic digestion reaction. Typically, this procedure involves overnight digestion of the protein or protein mixture prior to on-line ES/MS analysis.16,21,23,24,30 One approach to address this issue is the collisional activation of intact multiply charged intact proteins generated via electrospray ionization.31-38 It has been noted that multiple charging (17) Karty, J. A.; Lato, S.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1998, 12, 625-629. (18) Nelson, R. W.; Jarvik, J. W.; Taillon, B. E.; Tubbs, K. A. Anal. Chem. 1999, 71, 2858-2865. (19) Xiang, F.; Anderson, G. A.; Veenstra, T. D.; Lipton, M. S.; Smith, R. D. Anal. Chem. 2000, 72, 2475-2481. (20) Patterson, S. D.; Thomas, D.; Bradshaw, R. A. Electrophoresis 1996, 17, 877-891. (21) Figeys, D.; Corthais, G. L.; Gallis, B.; Goodlet, D. R.; Durcet, A.; Corson, M. A.; Aebersold, R. A. Anal. Chem. 1999, 71, 2279-2287. (22) Gygi, S. P.; Han, D. K. M.; Gingras, A. C.; Sonenberg, N.; Aebersold, R. Electrophoresis 1999, 20, 310-319. (23) McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R. Anal. Chem. 1997, 69, 767-776. (24) Figeys, D.; Gygi, S. P.; McKinnon, G.; Aebersold, R. Anal. Chem. 1998, 70, 3728-3734. (25) Li, J.; Thibault, P.; Bings, N. H.; Skinner, C. D.; Wang, C. Colyer, C.; Harrison, J. Anal. Chem. 1999, 71, 3036-3045. (26) Moore, R. E.; Licklider, L.; Schumann, D.; Lee, T. D. Anal. Chem. 1998, 70, 4879-4884. (27) Davis, M. T.; Lee, T. D. J. Am. Soc. Mass Spectrom. 1998, 9, 194-201. (28) Opiteck, G. J.; Lewis, K. C.; Jorgenson, J. W. Anderegg, R. J. Anal. Chem. 1997, 69, 1518-1524. (29) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1997, 69, 3177-3182. (30) Shevchenko, A,; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858. (31) Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 1998, 70, 3533-3544. (32) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991, 63, 24882499. (33) Senko, M. W.; Beu, S. C.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012808.

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facilitates the dissociation of high-mass ions, thereby allowing for the determination of structural information via the analysis of the dissociation products. For example, structurally diagnostic ions from dissociation of multiply charged proteins as large as bovine albumin (66 kDa) have been generated with commonly employed instruments, such as the triple quadrupole mass spectrometer.32 While multiple charging of parent ions has desirable consequences, it adds the complication that product ion charge states may vary from unity up to that of the parent ion. The product ion spectrum is therefore typically composed of ions of varying mass and charge. One approach to dealing with the product ion interpretation problem relies on the measurement of the massto-charge spacings between two or more ions related in some fashion to the species of interest.39,40 Recent studies in our laboratory based on the use of ion/ion proton-transfer chemistry subject the entire product ion population to ion/ion reactions, thereby leading to a product ion spectrum where singly charged ions dominate so that ambiguities in charge-state determination are minimized.31,35,37 Another major benefit of the collisional activation of intact protein ions is the presence of sequence-informative fragmentation regions adjacent to the major dissociation channels. In addition to charge-state reduction, the ion/ion reaction process effectively shifts the mass-to-charge ratio of these sequence-informative product ions (from the predominantly singly charged chemical background noise) to a region of higher mass to charge, where the signal-to-noise ratio is significantly greater. These sequenceinformative product ions derived from collisional activation of intact proteins are analogous to the “sequence tags” described by Mann and Wilm,41 generated from the collisional activation of proteolytic digest fragments. An analogous approach employing FTICR tandem mass spectrometry by McLafferty and co-workers42 for intact proteins, ranging in size from 8 to 43 kDa, demonstrated that the sequence tag approach is successful with a (2 Da mass restriction. This result suggests that tandem mass spectrometry (MS/MS) data acquired at lower mass accuracy with other instrumentation (i.e., quadrupole ion trap) could also be used for sequence tag retrievals. It is therefore conceivable that the “sequence tags” generated from collisional activation and ion/ ion reactions of intact protein ions in the quadrupole ion trap could be used to rapidly identify (by circumventing the proteolytic digestion step) a target protein of interest from a complex mixture.42 One area where this approach would be applicable is in the detection of viruses from complex biological matrixes. Bacte(34) Valaskovic, G. A.; Kelleher, N. L.; McLafferty F. W. Science 1996, 273, 1199. (35) Stephenson, J. L., Jr.; Cargile, B. J.; McLuckey, S. A. Rapid Commun. Mass Spectrom. 1999 13, 2040-2048. (36) Light-Wahl, K. J.; Loo, J. A.; Edmonds, C. G.; Smith, R. D.; Witkowska, H. E.; Shackleton, H. L.; Wu, C. S. S. Biol. Mass Spectrom. 1993, 4, 557. (37) Schaaff, T. G.; Cargile, B. J.; Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 2000, 72, 899-907. (38) Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler F. W., III; McLaferty, F. W. J. Am. Soc. Mass Spectrom. 1993, 4, 557. (39) Henry, K. D.; McLafferty, F. W. Org. Mass Spectrom. 1990, 25, 490-492. (40) Senko, M. W.; Beu, S. C.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1993, 4, 828-830. (41) Mann, M.; Wilm. M. Anal. Chem. 1994, 66, 4390-4399. (42) Mortz, E.; O′Connor, P. B.; Roepstorf, P.; Kelleher, N. L.; Wood, T. D.; McLafferty, F. W.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 82648267.

riophage MS2 is an icosahedral virus that is specific to bacteria that contain the F plasmid. This single-stranded RNA virus contains four genes that encode the coat protein (present at 180 copies/virion), an RNA-dependent polymerase, a lysis protein, and an assembly protein A (1 copy/virion) needed for the attachment of the virion to the male-specific pili of the host bacterium.43 Therefore, the coat protein is a useful marker for the detection of a virus in a complex bacterial lysate, due to the large number of copies of coat protein per virion (i.e., 180, MW ) 13 728). Fenselau et al.44 reported the direct analysis of viral capsid proteins (MS2, TMV-U2, and equine encephalitis TRD) via MALDI mass spectrometry; however, applications that involve direct infusion of complex mixtures using electrospray ionization are rare. We report here such a strategy for the identification of bacteriophage MS2 in an E. coli lysate. Thus, using sequence tags generated via collisional activation of the multiply charged ions of the intact viral coat protein in a complex matrix (with subsequent ion/ion protontransfer reactions to produce readily interpreted singly charged product ion mass spectra), the presence of the MS2 virus can be easily detected via database searching. EXPERIMENTAL SECTION The host bacterial strain Hfr+ E. coli (male specific) was obtained from American Type Culture Collection (ATCC No. 15669; Rockville, MD). Cultures were grown in 50-mL batches at 37 °C under aerobic conditions. The MS2 broth was prepared as described by Davis and Sinsheimer.45 Bacteriophage MS2 was also obtained from American Type Culture Collection (ATCC No. 15597-B1). Inoculation of the Hfr+ E. coli with bacteriophage MS2 was performed by the following procedure from Davis and Sinsheimer.45 The Hfr+ strain of E. coli was grown overnight in MS2 broth (16-18 h). Then two drops of Hfr+ E. coli were added to 50 mL of broth and grown to an optical density (OD) of 0.150.20 at 600 nm to ensure exponential growth conditions (Shimadzu UV-2101PC, dual-channel scanning UV-visible spectrophotometer; Columbia, MD). A small volume of bacteriophage MS2 stock suspension was added to the 0.15-0.20 OD culture for inoculation purposes. The culture was allowed to grow aerobically for 2-3 h at 37 °C. The end point for bacteriophage MS2 growth was observed when the culture changed from a cloudy dark yellow solution (0.15-2.0 OD) to a clear ivory-yellow (which closely resembled the original MS2 broth without any bacterial growth).44,45 The titers for the MS2 stock solutions ranged between 2 × 1010 and 9 × 1010 plaque-forming units/mL. Stock suspensions of bacteriophage MS2 were prepared by centrifuging the cultures in 50-mL conical centrifuge tubes at 5000g for 20 min using a Haraeus Biofuge 22R tabletop centrifuge (Haraeus Instruments; South Plainfield, NJ). Sample stock suspensions were prepared further by passing the supernatant through a 100 kDa molecular weight cutoff spin column (Millipore Corp.; Bedford, MA) at 3500g, to remove salts and other cellular debris. The resulting mixture of Hfr+ E. coli proteins and bacteriophage MS2 virions were concentrated to 1 mL (from the original 50-mL (43) Brock, T. D.; Madigan, M. T.; Markinko, J. M.; Parker, J. Biology of Microorganisms, 7th ed.; Prentice-Hall: Englewood Cliffs, NJ, 1994; Chapter 6. (44) Thomas, J. J.; Falk, B.; Fenselau C.; Jackman, J.; Ezzell, J. Anal. Chem. 1998, 70, 3863-3867. (45) Davis, J.; Sinsheimer, R. J. Mol. Biol. 1963, 6, 203-207.

culture) as a result of the ultrafiltration process. To dissociate the bacteriophage MS2 coat protein from its single-stranded RNA, acetic acid (Aldrich Chemical Co., Milwaukee, WI) was added to a final concentration of 66%.44,46 The resulting electrospray solutions were analyzed directly, diluted in 66% acetic acid, where concentrations ranged from 1:40 to 1:50 of the original 1 mL of stock. All solutions were directly infused at a flow rate (electroosmotic flow) between 20 and 40 nL/min through a home-built nanospray ion source. The source consisted of an x, y, z position manipulator from Newport Corp. (Irvine, CA), which was used to position a microelectrode holder from World Precision Instruments (Sarasota, FL). The original platinum wire from the microelectrode holder was replaced with a stainless steel wire (45-mm length, 0.1-mm i.d.), which was then inserted into a (1.5-mm-o.d., 0.86mm-i.d.) borosilicate glass capillary containing several microliters of sample solution. The connector on the microelectrode holder was then tightened to ensure needle stability. The glass capillary was pulled (∼5-µm tip) via a Sutter Instruments (Novato, CA) Flaming/Brown micropipet puller. The sample was loaded (1-3 µL) using an Eppendorf pipet with gel-loading tips. The electrospray needle voltage was optimized between 0.9 and 1.4 kV. The experiments were performed using a Finnigan MAT ion trap mass spectrometer (ITMS) modified for electrospray to allow for ion injection through an end-cap electrode47 and glow discharge for ion injection through a hole in the ring electrode.48 All experiments were controlled by ICMS software.49 A detailed description of the ion/ion reaction experiment including ion injection, desolvation, anion formation, ion activation, mutual storage/reaction time, and mass analysis scan can be found in the literature.31,35,37,48 Ion activation was performed using single-frequency resonance excitation.50,51 In all cases, parent ions were activated at a qz value of 0.205 (z-dimensional fundamental frequency of motion of ∼80 kHz). The best product ion peak shapes were observed using low resoanace excitation amplitudes (130-500 mV) and long activation times (250-500 ms). Furthermore, there is less likelihood for parent ion ejection at lower amplitudes. These observations are expected based on the slow heating nature of the ion trap collisional activation event.52,53 Details concerning ion activation at long activation times and low amplitudes have been described previously.31,35,37 The mass-to-charge scale (for low- and high-mass operation) was calibrated using the known molecular weight of the bacteriophage MS2 coat protein. Electrospray data (up to m/z 3600) from the +12 to +7 charge states and ion/ion reaction data (to encompass m/z 13729) from the +4 to +1 charge states of the bacteriophage MS2 coat protein provided the appropriate calibra(46) Fraenkel-Conrat, H. Virology 1957, 4, 1-4. (47) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1295. (48) Stephenson, J. L., Jr.; McLuckey, S. A. Int. J. Mass Spectrom. Ion Processes 1997, 162, 89-106. (49) ICMS software provided by N. Yates and the University of Florida. (50) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, G. C.; Todd, J. F. J. Anal. Chem. 1987, 59, 1677-1685. (51) Johnson, J. V.; Yost, R. A.; Kelley, P. E.; Bradford, D. C. Anal. Chem. 1990, 62, 2162-2172. (52) Goeringer, D. E.; McLuckey, S. A. J. Chem. Phys. 1996, 104, 2214-2221. (53) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 32, 461-474.

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tion data for the low- and high-mass ranges. This internal calibration was verified using electrospray and ion/ion reaction data from a 2 pmol/ul solution of myoglobin. The molecular weight of the bacteriophage MS2 coat protein was independently verified by acquiring electrospray mass spectra with a PE-Sciex API 365 triple quadrupole mass spectrometer calibrated with a solution of poly(propylene glycol). All spectra were acquired using the same voltages applied to the electron multiplier detector (1900 V) and conversion dynode (-5 kV). The post ion/ion reaction spectra derived from the product ions were typically the average of 1000-2000 scans. While mass determination of the product ions could be made with a factor of 50 or fewer scans, the data were extensively averaged in this work so that reliable sequence tags for database searching could be generated. For post ion/ion reactions derived from the parent ions, the average number of scans was 500-1000. The pre ion/ion reaction spectra represent an average of 100 scans. The mass accuracy associated with these measurements was 200-250 ppm. In the current instrumentation, mass accuracy is limited by undersampling of the peaks when the ion trap mass-to-charge range is extended beyond the usual upper limit of m/z 650. For the acquisition of post ion/ion reaction product ion spectra, the mass-to-charge range of the ion trap was extended by roughly a factor of 21. Mass accuracies as low as 20-30 ppm have been reported using ion traps without an undersampling limitation.54 The database searches were used by supplying protein masses with several mass accuracies ranging to as low as 100 ppm. While this mass accuracy is better than that used to acquire the data,54 it is not an unprecedented value for an ion trap and has therefore been included in the evaluation of the various search strategies. Database searching of the generated sequence tags was performed using two different on-line search engines. These include the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL (http:// prowl.rockefeller.edu/MSDB-html/)55 and the TagIdent tool from the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (http://www.expasy.ch).56 The SWISS-PROT database (release 38, 4/27/00, 85661 entries) was used by the ProteinInfo and TagIdent programs. All information including sequence tags and coat protein molecular weight were entered manually into both programs. RESULTS AND DISCUSSION Simplifying Mass Spectra of Complex Protein Mixtures. In Figure 1a is shown the electrospray mass spectrum of a 1:50 dilution of the original stock suspension of an MS2 infected E. coli extract. A prominent charge-state distribution ranging from +12 (m/z 1145) to +7 (m/z 1962) is clearly evident, where each of the peaks has a high enough signal-to-noise ratio to be detected over the large baseline rise associated with the E. coli extract. A voltage gradient of 170 V was established between the front plate of the electrospray interface and the first tube lens. This voltage gradient (through increased collision energy in the interface region) aids in the desolvation and removal of adduct species prevalent with the analysis of MS2 coat protein via mass spectrometric techniques. By employing traditional deconvolution (54) Williams, J. D.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1992, 6, 524527.

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Figure 1. (a) Electrospray ion trap mass spectrum of an MS2 infected E. coli sample. The data represent a 1:50 dilution of the original stock extract solution (concentrated to 1 mL during the isolation procedure) obtained from a 50-mL culture. (b) Ion/ion reaction mass spectrum (100-ms reaction time) of data from the electrospray experiment in (a). (c) Ion/ion reaction mass spectrum derived from the isolated +9 charge state of the coat protein found in (a).

algorithms, a value of 13 728 ( 0.4 is obtained for the molecular weight.57 Although the molecular weight value obtained from the deconvolution algorithm and the a priori knowledge that the E. (55) Fenyo, D.; Zhang, W.; Chait, B.; Beavis, R. C. Anal. Chem. 1996, 68, 721A726A. (56) Appel, R. D.; Bairoch, A.; Hochstrasser, D. F. Trends Biochem. Sci. 1994, 19, 258-260.

coli ribosomes were used to translate primarily viral proteins suggest that the charge-state distribution is that of the coat protein of bacteriophage MS2, the observed mass accuracy alone cannot be used to unequivocally identify this protein.58 Additional information in the form of a peptide mass fingerprint or the generation of a sequence tag is necessary for unambiguous identification.59,60 Although peptide mass fingerprinting is an effective technique for the identification of proteins, a great deal of time and effort are still required in the proteolytic digestion step (and possibly a chromatographic cleanup step). Since the objective of our experiment is to minimize sample manipulation steps, sequence tag generation via the collisional activation of intact multiply charged protein ions followed by subsequent charge-state reduction via ion/ion proton-transfer chemistry can simplify the analysis by placing additional burden on the mass spectrometer. In Figure 1b is shown the ion/ion reaction mass spectrum of the E. coli extract (containing bacteriophage MS2) after the reaction of the entire multiply charged ion population for 100 ms with perfluoro-1,3-dimethylcyclohexane (PDCH) anions. The predominant peaks in this mass spectrum represent the charge reduction products ranging from the +4 to the +1 charge state of the viral coat protein. The average MS2 ion charge state could be varied arbitrarily via selection of the ion/ion reaction time. However, this ion/ion reaction mass spectrum is also characterized by an elevated baseline between m/z 3000 and 8000 (chemical noise and various other peaks associated with the E. coli extract). The presence of an elevated baseline along with clearly apparent but unidentified peaks can complicate the interpretation of singly charged product ion spectra (produced via ion/ion chemistry) needed for sequence tag generation. In order for the ion/ion strategy to be effective for generating sequence tags using intact proteins, the appearance of chemical noise at moderate to high m/z values (in the case of the MS2 coat protein approximately m/z 3000-13800) should be minimized. Isolation of a single charge state followed by subsequent ion/ion reaction can significantly reduce the background noise level and number of interfering peaks associated with ion/ion reactions of crude extracts. In Figure 1c, the ion/ion reaction mass spectrum from the isolated +9 charge state of the viral coat protein is shown. When compared to the ion/ion reaction data generated from the charge-state reduction of the entire mixture in Figure 1b, a significant reduction in baseline chemical noise is observed. However, Figure 1c also shows a series of low-abundance signals which correspond to the overlapping m/z value associated with the isolated +9 charge state of the viral coat protein. These post ion/ion reaction “noise/ unwanted protein peaks” are labeled according to their starting/ original charge-state value when they directly overlap with m/z 1526 (+9 charge state of the viral coat protein). For example, the peaks labeled [+8], [+8/2], and [+8/3] all correspond to ions arising from the +9 species in the original isolated ion population at approximately m/z 1526. (57) Smith, C. R., Gandy, Jr., W. T., Eds.; Maximum Entropy: Bayesian Methods; Reidel: Dordrecht, 1985. (58) Mann, M.; Hojrup, P.; Roepstorff Biol. Mass Spectrom. 1993, 22, 338-345. (59) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5011-5015. (60) Yates, J. R., III; Speicher, S.; Griffin, P. R.; Hunkapiller, T. Anal. Biochem. 1993, 214, 397-408.

A further reduction of chemical background noise for the collisional activation/charge-state reduction process is accomplished by performing a double-isolation experiment (data not shown). In this experiment, the first step involves isolation of the next highest charge state of interest, followed by a short ion/ion reaction time (∼30 ms) to produce a charge-state distribution where the abundance of the next lowest charge state is maximized. This experiment separates the starting ion population of interest from chemical background noise and other interfering peaks by lowering the charge state of the isolated precursor ion by one. This new precursor ion can then be isolated, and subsequent MS/ MS experiments are then performed with a small amount of starting background noise. Deriving Structural Information from Protein Product Ion Spectra. Figure 2 compares the various ways in which spectra of dissociation products derived from the [M + 9H]9+ precursor ion can be displayed. In each experiment, a double isolation was performed where the +10 charge state of the MS2 coat protein was isolated and allowed to react to primarily the +9 charge state. Next, the +9 charge state was isolated and subsequent collisional activation was performed on the intact protein. From this point, ion/ion reaction experiments were carried out on the product ion population. The data shown in Figure 2a are the MS/MS spectrum of the intact protein with no post ion/ion reaction period. Due to the potential for overlap of ions with the same mass-to-charge ratio but different charge (e.g., the +3 charge state of y37 and the +1 charge state of y13 peaks at m/z 1275), interpretation of Figure 2a is highly ambiguous. A far less ambiguous interpretation could be made after subjecting the entire product ion population to ion/ ion reactions for 105 ms as shown in Figure 2b. From Figure 2b, the majority of the high-mass fragments observed correspond to b-type ions arising from cleavages near the C-terminal end of the intact +9 charge state. The most intense ions in the spectrum b118 (Ile118-Pro119), b116 (Asn116-Pro117), and b92 (Ile92-Pro93) represent cleavage on the N-terminal side of proline, a phenomenon that has been observed previously with activation/dissociation studies of other intact protein ions.31,35,37,61 The singly charged y37 ion shown in Figure 2b is the complement to the b92 ion. The presence of the complementary y-type ions y11 (Ile118-Pro119) and y13 (Asn116Pro117) can be verified by performing an ion/ion reaction experiment with the instrument scan range set to the same parameters used for the conventional electrospray ionization experiment (m/z 600-2200, seen in Figure 2a). As observed with all ion/ion reaction experiments, the reactions proceed as a function of the square of the cation charge state.62 Therefore, for a fixed ion/ion reaction time of 105 ms, all the highly charged b-type ions seen in Figure 2a will react at a much faster rate than their complementary singly charged y-type ion counterparts. The end result shown in Figure 2c (scanning the same mass range as shown in Figure 2a) confirms the presence of both the singly charged y11 and y13 peaks, along with various other singly charged species originally present as singly charged ions derived from collisional activation of the [M + 9H]9+ precursor ion. Conspicuously absent from this spectrum are prevalent fragment ions associated with cleavage on the C-terminal side of aspartic and glutamic acid (61) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1993, 65, 425-438. (62) Stephenson, Jr. J. L.; McLuckey, S. A. J. Am. Chem. Soc. 1996, 118, 73907397.

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Figure 2. (a) MS/MS data on the +9 charge state (double isolation experiment) of the MS2 viral coat protein. (b) Post ion/ion reaction MS/MS data (high mass) derived from the +9 charge state (double isolation experiment) of the MS2 viral coat protein from (a). The fragmentation at adjacent residues which occurs in the b116 (Asn116-Pro117) ion region is shown in the inset. (c) Post ion/ion reaction MS/MS data (low mass) derived from the +9 charge state (double isolation experiment) of the MS2 viral coat protein from (a). The scan range for this experiment is the same as in (a).

residues (another previous observation we have noted with activation/dissociation of intact protein ions31,35,37). In the case of the +9 charge state, the only cleavages linked with the C-terminal side of acidic residues (out of nine possible cleavage points, Asp11,17,100,114 and Glu31,63,76,89,102) are the y40 (Glu89-Leu90) and b114 1282

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(Asp114-Gly115) ions, which are present at low abundance (where the superscripts to the right of the amino acid residue refer to the position of that residue in the protein counting from the N-terminus). Although three (Pro93,117,119) of the six (Pro22,65,78,93,117,119) possible cleavages on the N-terminal side of proline are observed for the +9 charge state, it is apparent that other factors are also important in governing protein ion dissociation. The appearance of a series of product ions due to dissociation at adjacent residues is only observed in the region of the b116 (Asn116-Pro117) ion derived from the +9 charge state (see inset in Figure 2b). Here the observed dissociation at adjacent residues, from which sequence tags can be generated, can be used to aid in protein identification. From the product ion sequence ranging from the b108-b115 ions shown in Figure 2b, a partial amino acid sequence of Gln-Gly-Leu-Leu-Lys-Asp-Gly-Asn is obtained. However, for the slow-heating collisional activation experiment typical of the quadrupole ion trap, differentiation of the side-chain fragmentation between the isomers of leucine and isoleucine (residual mass 113) is not possible. In addition, differentiation between lysine (residual mass 128.095) and glutamine (residual mass 128.058) is problematic (without acetylation of the free primary amine group of lysine) due to the limited resolving power associated with quadrupole instrumentation. Therefore, the partial amino acid sequence should read as {Gln /Lys}-Gly-{Leu/Ile}{Leu/Ile}-{Lys/Gln}-Asp-Gly-Asn, where {Gln/Lys} and {Leu/ Ile} pairs are in braces, indicating a choice of two amino acids for a given position in the partial sequence. To determine the presence of other unique sequence tags for the MS2 coat protein, MS/MS experiments were performed on several other charge states. In Figure 3a is shown the post ion/ ion MS/MS spectrum of the +8 charge state. As with the +9 charge state, the major peaks observed in the MS/MS spectrum of the +8 charge state are b118 (Ile118-Pro119), b116 (Asn116-Pro117), and b92 (Ile92-Pro93), which correspond to cleavages on the N-terminal side of proline. However, the abundance of the complementary ion pair of b92 and y37 (Ile92-Pro93) is greatly increased over that observed in the +9 charge state. More extensive fragmentation at adjacent residues is observed in the region of the b116 ion for the MS/MS data of the +8 charge state than was seen in the +9 charge state. The b-type ion series ranging from b104 to b116 yielded an amino acid sequence tag of 12 residues Val-{Lys/Gln}-Ala-Met-{Gln/Lys}-Gly-{Leu/Ile}{Leu/Ile}-{Lys/Gln}-Asp-Gly-Asn, where an additional four cleavages from b104 to b107 (Val-{Lys/Gln}-Ala-Met) on the N-terminal side of the b116 are observed. In addition, fragmentation at adjacent residues occurs in the b122 ion region for the MS/MS data of the +8 charge-state ion. This small series of cleavages b120-125 (Ala{Ile/Leu}-Ala-Ala-Asn) at adjacent residues is of particular interest since the prominent peak in the series, b122, represents cleavage between two alanine residues and not the usual N-terminal proline or C-terminal acid residue fragmentation typically associated with the most intense ions derived from the MS/MS of intact proteins. The product ion spectrum (post ion/ion reaction) derived from the MS/MS of the +7 charge state of MS2 coat protein is shown in Figure 3b. The dominant peaks associated with this spectrum are the b92/y37 complementary pair associated with bond cleavage on the N-terminal side of Pro93. Noticeably reduced in abundance in this MS/MS spectrum are the major fragments related to

Table 1. Sequence Tags Generated for MS/MS of the +9, +8, and +7 Charge States of the MS2 Coat Protein charge state +9 +8 +7

sequence tagsa,b [Q/K]G[L/I][L/I][K/Q]DGN V[K/Q]AM[Q/K]G[L/I][L/I][K/Q]DGN [L/I]T[I/L]

A[I/L]AAN FATNSD

a Amino acid residues in brackets indicate an ambiguous assignment due to the same nominal mass values for both components. b Amino acids in boldface type within the brackets represent the actual sequence of the bacteriophage MS2 coat protein.

Figure 3. (a) Post ion/ion reaction MS/MS data derived from the +8 charge state (double isolation experiment) of the MS2 viral coat protein. The fragmentation at adjacent residues which occurs in the b116 (Asn116-Pro117) ion region is shown in the inset. (b) Post ion/ion reaction MS/MS data derived from the +7 charge state (double isolation experiment) of the MS2 viral coat protein. The fragmentation at adjacent residues which occurs in the b92 (Ile92-Pro93) ion region is shown in the inset.

cleavage of (Ile118-Pro119) and (Asn116-Pro117) representing the b118 and b116 ions, respectively. Also, the large number of consecutive cleavages at adjacent amino acid residues previously observed for the +8 and +9 charge states in the b116 ion region are not observed with the +7 charge state. Primarily, cleavages at every other amino acid residue are seen in this region with the following ion sequence of b111, b112, b114, b116, b118. However, sequence tag information can be obtained from the b92 ion region. The threeamino acid sequence tag {leu/Ile}-Thr-{Ile/Leu} corresponding to the b89-b92 series, and the six-amino acid sequence tag Phe-Ala-Thr-Asn-Ser-Asp corresponding to the b94-b100 series is shown in the expanded view inset of Figure 3b. Examination of the peaks in the complementary y37 region produced no additional sequence tag information since none of the observed peaks could be correlated with known protein fragment assignments (a,b,c,w,x,y,z). A summary of the sequence tag information generated from the three MS/MS ion/ion reaction spectra of the +9, +8, and +7

charge states is shown in Table 1. The sequence tag data from this table was used in the database searches in various forms including charge-state-specific tags and tags searched with the known molecular weight of the coat protein. Since the fragment ions generated from the MS/MS experiment cannot be categorized a priori as b- or y-type ions from the product ion spectrum alone (assuming an unknown protein), all sequence tags were searched in the forward and reverse directions. Database Interrogation Using Information Derived from Product Ion Spectra. To evaluate the data produced from the collisional activation of intact multiply charged protein ions, two specific web-based identification programs were employed. Using the ProteinInfo program from the PROWL website,55 each individual sequence tag from each of the three charge states was used to search the SWISS-PROT database. Brackets were employed to specify the indistinguishable pairs of amino acids [IL] and [KQ]. The advantages of this search routine are the ability to identify proteins in the presence of unknown posttranslational modifications, insensitivity of the algorithm to amino acid substitutions (e.g., single nucleotide polymorphisms), and or possible errors in the sequence database. Also, all sequence tags were searched in the reverse direction (see values in parentheses in Table 2) since it is unknown whether the tag consists of a b-or y-type ion series. In Table 2 are shown the results of the sequence tag searches. For the +9 charge state, the sequence tag [Q/K]G[L/I][L/I][K/ Q]DGN yielded three matching proteins of which one was the coat protein for the MS2 bacteriophage. The other two matches were the coat proteins of bacteriophage F2 and R17. By further examination of these three database entries with the molecular weight qualifier of 13 728, the mass accuracy of the measurement can be used to rule out the F2 coat protein (MW ) 13 709.0). On the other hand, the R17 coat protein with a molecular weight of 13 727.5 cannot be entirely eliminated as a possibility due to the 1 Da difference between it and the MS2 coat protein. It was found that this 1 Da difference was due to the substitution of Asp17 with Asn17, and all other amino acid residues were found to be the same in both the MS2 and R17 coat proteins. Correspondingly, a reverse search of this sequence tag yielded no matches in the database. This unusually high degree of protein homology does present a problem for the intact protein MS/MS approach, in that very good mass accuracies are needed for the higher mass ions associated with ion/ion reactions of product ion spectra. The chances of distinguishing this subtle difference between the MS2 and R17 coat protein are considerably better using proteolytic digestion and peptide mapping, where the fragment masses Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

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Table 2. Database Search Results from the ProteinInfo Program of PROWL charge state +9 +8

sequence

tagsa,b

no. of matching proteinsc,d

no. of matching proteins with a MW qualifierc,d

3 (0) 3 (0) 29 (83) g100 (g100) 3 (1)

2 (0) 2 (0) 2 (0) n/ae 2 (0)

[Q/K]G[L/I][L/I][K/Q]DGN V[KQ]AM[Q/K]G[L/I][L/I][K/Q]DGN A[I/L]AAN [L/I]T[I/L] FATNSD

+7

a Amino acid residues in brackets indicate an ambiguous assignment due to the same nominal mass values for both components. b Amino acids in boldface type within the brackets represent the actual sequence of the bacteriophage MS2 coat protein. c Results for SWISS-PROT database. d Results of searching the sequence tag in reverse order, values in parentheses. e Not available.

Table 3. Database Search Results from the TagIdent Program from the ExPASY Molecular Biology Server database charge state

sequence taga

search qualifier

0.2%

+9

QGLLKD

MW tage MW tage MW tage MW tage MW tage MW tage MW tage

80 3/3 80 3/3 80 3/3 80 3/3 80 4/4 80 21/10 80 3/3

LLKDGN +8

VKAMQG LLKDGN AIAAN

+7

LTI FATNSD

SWISS-PROTb mass accuracyd 0.1% 0.05% 0.02% 41 2/2 41 2/2 41 2/2 41 2/2 41 3/3 41 13/6 41 2/2

19 2/2 19 2/2 19 2/2 19 2/2 19 2/2 19 8/4 19 2/2

7 2/2 7 2/2 7 2/2 7 2/2 7 2/2 7 2/2 7 2/2

0.01%

0.2%

6 2/2 6 2/2 6 2/2 6 2/2 6 2/2 6 2/2 6 2/2

138 1/0 138 2/0 138 0/0 138 2/0 138 1/0 138 24/10 138 0/0

TrEMBLc mass accuracyd 0.1% 0.05% 0.02% 81 1/0 81 1/0 81 0/0 81 1/0 81 1/0 81 13/6 81 0/0

40 1/0 40 1/0 40 0/0 40 1/0 40 1/0 40 6/2 40 0/0

17 0/0 17 0/0 17 0/0 17 0/0 17 1/0 17 2/1 17 0/0

0.01% 8 0/0 8 0/0 8 0/0 8 0/0 8 0/0 8 1/1 8 0/0

a Sequence tags longer than the six amino acid maximum allowed by TagIdent were split into two separate tags. For the +8 charge state, the tag VKAMQGLLKDGN was split into VKAMQG and LLKDGN. For the +9 charge state, the tag QGLLKDGN was split into QGLLKD and LLKDGN, which represents a two-amino acid shift across the partial sequence of 8 amino acids. b SWISS-PROT database search with entries. c TrEMBL database search with entries. d Mass accuracy values used in the program translate to the following ∆s: 0.2% with ∆ ) 27 Da, 0.1% with ∆ ) 13 Da, 0.05% with ∆ ) 6 Da, 0.02% with ∆ ) 2 Da, and 0.01% with ∆ ) 1 Da. e Sequence tags searched for all permutations. The first number indicates the number of matches regardless of the sequence tag permutations. The second number in boldface type indicates the number of matches with the sequence tag in the correct order.

produced can be discerned at the 1 Da level. The probability that the three matches are nonrandom can be calculated by the method of Mann and Wilm.41 Thus, for a sequence tag of eight amino acids (considering no tryptic peptides or mass values), the probability that the sequence is nonrandom is 96.4% for the SWISSPROT database with ∼3.1 × 107 amino acids. In the case of the +8 charge state, there are two sequence tags, the 12- and 5-amino acid sequences V[K/Q]AM[Q/K]G[L/ I][L/I][K/Q]DGN and A[I/L]AAN, respectively. Search results for the 12-amino acid tag give the same three matches as were found for the amino acid tag derived from the +9 charge state. As with the +9 charge state, it would be extremely difficult to distinguish the MS2 and R17 coat proteins. For the reverse search, no matches to the database were obtained, and the calculated probability of a nonrandom match approached 100%. The fiveamino acid tag produced 29 matches. However, all but two of these could be eliminated using the molecular weight qualifier. Interestingly, the reverse search produced 83 matches, but by employing the molecular weight qualifier, all 83 could be excluded from consideration. The probability that this sequence is nonrandom is infinitesimally small. 1284 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

The two amino acid tags for the +7 charge state have the smallest combination of sequence tag information of all the charge states. The results from the first tag [L/I]T[I/L], searched in both the forward and reverse directions, exceeded 100 matches, which is the limit of the ProteinInfo program. The second tag of FATNSD again yielded the same three matches observed with the 8- and 12-amino acid tags from the +9 and +8 charge states, with the conclusion that distinguishing between the coat proteins of MS2 and R17 would be extremely difficult without peptide mapping. The reverse search for the FATNSD tag produced one match. However, the molecular weight of the entry was 66 000 and was therefore eliminated from consideration. The observed probability of a nonrandom match for the [L/I]T[I/L] is infinitesimally small while that of the FATNSD tag is on the order of 38.8%. A summary of the results from the TagIdent program from the ExPASy molecular biology server56 is shown in Table 3. For this program, sequence tags of only six amino acids in length can be used. For database search purposes, the [L/I]T[I/L] and FATNSD tags derived from the +7 charge state and the A[I/L]AAN tag from the +8 charge state could be entered directly into the program. The 12-residue tag V[K/Q]AM[Q/K]G[L/I][L/I]-

[K/Q]DGN of the +9 charge state was split into two 6-residue tags composed of V[K/Q]AM[Q/K]G and [L/I][L/I][K/Q]DGN, while the 8-residue tag [Q/K]G[L/I][L/I][K/Q]DGN was split into two sequences of [Q/K]G[L/I][L/I][K/Q]D (amino acids 1-6) and [L/I][L/I][K/Q]DGN (amino acids 2-8). In addition, the program does not allow for an either/or qualifier in the case of leucine/isoleucine and lysine/glutamine. Therefore, the exact sequence was entered into the program with a priori knowledge of the leucine/isoleucine and lysine/glutamine pair. To accommodate reverse searches, all possible permutations of the tags were compared against the database. One aspect of this program that lends itself to the intact protein ion/ion approach for protein identification is the fact that molecular weight (with varying degrees of mass accuracy) can be directly incorporated into the search routine. Mass accuracy values of 0.2, 0.1, 0.05, 0.02, and 0.01% were used with both the SWISS-PROT and TrEMBL databases. In the SWISS-PROT searches, the number of matches returned for molecular weight qualifier were 80, 41, 19, 7, and 6 for accuracies of 0.2, 0.1, 0.05 0.02, and 0.01%, respectively (see Table 3). For the nonannotated/redundant TrEMBL entries, the matches retrieved were 138, 81, 40, 17, and 8 for the identical mass accuracies. The data shown for sequence tag matching for a corresponding mass accuracy is displayed as two values in Table 3 with the first representing all permutations of the tag and the second matching only the tag in the correct order (data shown in boldface type in Table 3). For the +7 charge-state tag of LTI (which was shown previously to be too general to use as a way to identify the protein by itself) coupled with an accurate enough mass measurement (0.02% or greater), the matches could again be narrowed to the coat protein of MS2 and R17 by searching the SWISS-PROT database. Searches against the TrEMBL database at an accuracy of 0.01% yielded one additional protein match, indicating that a more specific sequence would be needed for unambiguous identification. The more specific tag FATNSD (+7 charge state) showed that, even at a mass accuracy as low as 0.2%, the number of possible matches could be narrowed to three possibilities (the MS2, R17, and F2 coat proteins as discussed previously). The same results were also obtained for the LLKDGN tag derived from the MS/MS of the +9 and +8 charge states, the VKAMQG tag from the +8 charge state, and the QGLLKD tag from the +9 charge state. In each of the aforementioned cases, searches against the TrEMBL database yielded no additional matches for the exact sequence tag regardless of mass accuracy. For the AIAAN tag obtained from the MS/MS of the +8 charge state, the 0.2% mass accuracy gave four possible matches, and with a mass accuracy of 0.05% or greater could narrow the range of possibilities to the MS2 and R17 coat proteins. As with the previous example, searches against the TrEMBL database gave no additional matches.

CONCLUSIONS The data generated via MS/MS of intact proteins, followed by ion/ion reaction chemistry to identify product ions, appears to provide the criteria necessary for rapid identification of target proteins from real biological samples. By performing multiple isolation experiments with ion/ion chemistry, a sufficient number of unwanted ions with the same m/z value of the charge state of interest can be removed prior to the MS/MS experiment. This leads to the improved signal-to-noise ratios necessary for obtaining sequence tag information needed to search protein databases. The combination of several MS/MS experiments on a variety of charge states, followed by ion/ion reactions, can yield unique chargestate-specific fragmentation, which helps identify a given protein in the database with an increased confidence level. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via adjacent cleavages is highly dependent upon parent ion charge state. Although the sequence tag information provided from MS/ MS of intact proteins can be used with a variety of search engines, the approaches described here were specifically chosen to evaluate the usefulness of the quadrupole ion trap collisional activation experiment to generate appropriate sequence tag information for protein identification. For both the ProteinInfo and TagIdent programs, sequence tag information generated for the +7, +8, and +9 charge states could be used to correctly identify the MS2 coat protein. Further studies planned for improved mass resolution/accuracy, and development of data interpretation algorithms designed specifically for the slow heating collisional activation data generated by quadrupole ion traps, should provide increased performance for this methodology soon. ACKNOWLEDGMENT Quadrupole ES-MS instrumentation was provided through a Cooperative Research and Development Agreement with PerkinElmer Sciex Instruments CRADA ORNL96-0458. This research was sponsored by the Office of Research and Development (NN20, Chemical and Biological Non-Proliferation Program), U.S. Department of Energy, under Contract DE-AC05-00OR22725 with Oak Ridge National Laboratory, managed and operated by UTBattelle, LLC. B.J.C. acknowledges support through an appointment to the Professional Internship Program, administered by the Oak Ridge Institute for Science and Education. Received for review June 23, 2000. Accepted January 10, 2001. AC000725L

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