High-Resolution Multistage MS, MS2, and MS3 Matrix-Assisted Laser

Monitoring Protein Expression in Whole Bacterial Cells with MALDI ... as an Effective Tool for Investigating the Epitranscriptomics of Stress Response...
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Articles Anal. Chem. 1996, 68, 3718-3725

High-Resolution Multistage MS, MS2, and MS3 Matrix-Assisted Laser Desorption/Ionization FT-ICR Mass Spectra of Peptides from a Single Laser Shot Touradj Solouki, Ljiljana Pasˇa-Tolic´, George S. Jackson,† Shengheng Guan,† and Alan G. Marshall*,†

Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Florida State University, Tallahassee, Florida 32310

By combined and repeated use of sustained off-resonance irradiation (SORI) for ion dissociation, stored waveform inverse Fourier transform (SWIFT) waveforms for ion isolation, and ion axialization and remeasurement techniques, we obtain for the first time MS, MS2, and MS3 FT-ICR mass spectra from peptide ions (enzymatic digest products of horse cytochrome c) produced from a single laser shot. The successive fragmentation of gas-phase ions detected from the same initial batch of ions increases the sensitivity of analysis of trace amounts of biological samples in structural mass spectrometry, and fragment identification is facilitated by resolution of carbon-13 isotopic distributions. The method is illustrated by analyses of subfemtomole amounts of crudely purified samples of tryptic digest solutions of horse cytochrome c and bovine cytochrome c. The high-resolution primary ion mass spectrum, along with the collision-induced dissociation (CID) and MSn capabilities of FT-ICR, help to determine the primary amino acid sequence of the fragment ions beyond what is obtained from enzymatic digestion alone, without prior chromatographic separation and purification. Peptide Mapping and Sequencing. Peptide mapping,1 one of the most powerful and successful tools available for protein studies, is traditionally performed by digesting a protein with complementary endoproteases, separating the protein digest components by (reversed-phase) high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE), followed by sequencing of each separated peptide starting from the amino-terminus based on automated Edman degradation, and then matching the overlapping peptide sequences to obtain the full primary amino acid sequence of the protein. This process is typically very time consuming, requiring about a month even for a relatively small protein (∼20 000 Da). The alternative approach of sequencing proteins indirectly by DNA sequencing of their corresponding genes2,3 has generated voluminous primary struc† Members of the Department of Chemistry, Florida State University. (1) Ingram, V. M. Nature 1956, 178, 792-794. (2) Gilbert, W. Science 1981, 214, 1305-1313. (3) Sanger, F. Science 1981, 214, 1205-1211.

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ture data over the last decade. Given the large number of known protein primary amino acid sequences, it is now possible to identify a protein on the basis of mass spectrometric mapping of a protein digest, coupled with database searching of sequences with the detected masses. A typical procedure consists of enzymatic cleavage of the protein, followed by separation of components (i.e., HPLC or CZE) prior to ionization (fast atom bombardment, FAB; electrospray ionization, ESI; or matrix-assisted laser desorption/ ionization, MALDI) and mass analysis. Although classical Edman degradation remains the principal technique for sequence determination, the mass spectrometric approach is fast and sensitive. Moreover, for sequence analysis of oligopeptides containing blocked N-termini, posttranslational modifications, or unusual amino acids, mass spectrometry represents the only (or at least the best suited) analytical method. Although mass spectrometry-based amino acid sequencing has been available for a few decades,4,5 the introduction of MALDI6 and ESI7 techniques revolutionized this field by greatly extending the upper mass limit and lowering the detection limit. Furthermore, Chait and co-workers have introduced “ladder sequencing” of proteins, consisting of modified stepwise Edman degradation, followed by readout of the primary structure of the protein from a MALDI time-of-flight (TOF) low-resolution mass spectrum.8 Peptide sequencing has also been attempted by mass spectrometry alone: e.g., the MALDI-generated bradykinin molecular ion has recently been collisionally activated to obtain partial sequence from several stages (up to MS4) of mass analysis.9 FT-ICR MSn (n e 4) experiments have also been demonstrated on ESIgenerated multiply-charged protein and oligonucleotide ions.10-13 Apart from determination of primary structure of proteins, ESI (4) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111. (5) Biemann, K. The Utility of Mass Spectrometry for the Determination of the Structure of Peptides and Proteins: An Overview; McEwen, C. N., Larsen, B. S., Eds.; Marcel Dekker, Inc.: New York, 1990; pp 3-24. (6) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (7) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (8) Chait, B. T.; Wang, R.; Beavis, R. C.; Kent, S. B. H. Science 1993, 262, 89-92. (9) Huang, Y.; Pasa-Tolic, L.; Guan, S.; Marshall, A. G. Anal. Chem. 1994, 66, 4385-4389. (10) Wu, Q.; Van Orden, S.; Cheng, X.; Bakhtiar, R.; Smith, R. D. Anal. Chem. 1995, 67, 2498-2509. S0003-2700(96)00312-5 CCC: $12.00

© 1996 American Chemical Society

and/or MALDI mass spectrometry have been applied successfully to characterize noncovalently bound complexes,14-16 to examine the conformational relationship between solution and gas phase by use of standard tandem MS techniques and hydrogen/ deuterium exchange,17-22 and for bioaffinity characterization of enzyme-inhibitor complexes.23 Matrix-assisted laser desorption/ionization complements electrospray, because MALDI shows much higher tolerance toward common contaminants: e.g., salts, buffers, and detergents.24,25 Extensive purification and manipulation are therefore not required prior to MALDI mass analysis. In the 1980s, protein chemistry was based mainly on the combined use of HPLC and fast atom bombardment (FAB) ionization, coupled with double-focusing sector mass analyzers.4,5,26 Today, most MS-based peptide mapping and sequence analyses of proteins is based on MALDI time-of-flight instruments or ESI sources coupled with on-line or off-line LC/MS analysis. Moreover, MALDI-TOF mass spectrometry has been applied to analysis of peptides isolated from support-bound combinatorial libraries.27 However, MALDI-TOF mass resolving power is typically low (m/ ∆m < 1000, where m is ion mass and ∆m is the mass spectral peak width at a specified fraction of maximum peak height, e.g., 50%), and tandem mass spectrometry with reflectrons is problematic, especially over a wide mass range. Coupling of a MALDI source with a FT-ICR mass analyzer,9,28-37 on the other hand, offers potentially ultrahigh mass resolving power, precise mass measure(11) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Cornnor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (12) Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1994, 116, 4893-4897. (13) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012808. (14) Li, Y. T.; Hsieh, Y. L.; Henion, J. D.; Ganem, B.; Senko, M. W.; McLafferty, F. W. J. Am. Chem. Soc. 1993, 115, 8409. (15) Woods, A. S.; Buchsbaum, J. C.; Berg, J. M.; Cotter, R. J. Proceedings of the 43rd ASMS Conference on Mass Spectrometry & Allied Topics, Atlanta, GA, May 21-26, 1995; WPI237. (16) Smith, D. L.; Zhang, Z. Mass Spectrom. Rev. 1994, 13, 411-429. (17) Chowdhury, S. K.; Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1990, 112, 9012. (18) Winger, B. E.; Light-Wahl, K. J.; Rockwood, A. L.; Smith, R. D. J. Am. Chem. Soc. 1992, 114, 5897-5898. (19) Miranker, A.; Robinson, C. V.; Radford, S. E.; Aplin, R. T.; Dobson, C. M. Science 1993, 262, 896-900. (20) Suckau, D.; Shi, Y.; Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M., III; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 790-793. (21) Wagner, D. S.; Anderegg, R. J. Anal. Chem. 1994, 66, 706-711. (22) Anderegg, R. J.; Wagner, D. S.; Stevenson, C. L.; Borchardt, R. T. J. Am. Soc. Mass Spectrom. 1994, 5, 425-433. (23) Smith, R. D.; Bruce, J. E.; Chen, R.; Cheng, X.; Schuartz, B. L.; Anderson, G. A.; Hofstadler, S. A.; Gale, D. C., Proceedings of the 43rd ASMS Conference on Mass Spectrometry & Allied Topics, Atlanta, GA, May 21-26, 1995; p 682. (24) Beavis, R. C.; Chait, B. T. Anal. Chem. 1990, 62, 1836-1840. (25) Beavis, R. C.; Chaudhary, T.; Chait, B. T. Org. Mass Spectrom. 1992, 27, 156-158. (26) Protein Sequence Analysis by Tandem Mass Spectrometry; Hunt, D. F., Shabanowitz, J., Yates, J. R., Griffin, P. D., Zhu, N. Z., Eds.; Marcel Dekker, Inc.: New York, 1990; pp 169-195. (27) Youngquist, R. S.; Fuentes, G. R.; Miller, C. M.; Ridder, G. M.; Lacey, M. P.; Keough, T. Proceedings of the 43rd ASMS Conference on Mass Spectrometry & Allied Topics, Atlanta, GA, May 21-26, 1995; p 484. (28) Hettich, R. L.; Buchanan, M. V. Int. J. Mass Spectrom. Ion Processes 1991, 111, 365-380. (29) Nuwaysir, L. M.; Wilkins, C. L. Proc. SPIE-Appl. Spectrosc. Mater. Sci. 1991, 112-123. (30) Solouki, T.; Russell, D. H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 57015704. (31) Solouki, R.; Gillig, K. J.; Russell, D. H. Anal. Chem. 1994, 66, 1583-1587. (32) Castoro, J. A.; Ko ¨ster, C.; Wilkins, C. Rapid Commun. Mass Spectrom. 1992, 6, 239-241. (33) Buchanan, M. V.; Hettich, R. L. Anal. Chem. 1993, 65, 245A-259A.

ment, and multistage MSn in a single instrument.38-49 FT-ICR mass spectrometers interfaced with either a liquid secondary ion mass spectrometry (SIMS) ion source26 or with an ESI source10,13,50 have been successfully applied to peptide sequence analysis. Simultaneous detection of all ions permits a significant reduction in sample consumption. Also, since a Penning trap is an ion storage device, it permits accumulation of ions produced in small abundance from a small amount of sample. Ion Remeasurement. Nondestructive FT-ICR detection allows for multiple detection (or remeasurement)51,52 of ionized biomolecules with dramatically improved sensitivity. The combined use of azimuthal quadrupolar excitation and collisional damping53 allows for initially detected ions to be brought back near the central axis of the trap and remeasured. A signal-tonoise ratio enhancement factor as large as n1/2, where n is the number of summed time domain data sets, can be achieved by such remeasurement.54 Laude and co-workers employed ion remeasurement techniques to observe real-time charge stripping of electrosprayed biomolecules.55 Recently, it has been shown that ion multiple remeasurements combined with sample deposition onto a small indentation on the probe tip allow MALDI FTICR mass analysis of low attomole amounts of peptides and phospholipids.52 MALDI FT-ICR MS sensitivity compares favorably to that previously reported for MALDI-TOF56,57 or an ESI source coupled with any of several mass analyzers.58-63 Dissocia(34) Ko ¨ster, C.; Castoro, J. A.; Wilkins, C. L. J. Am. Chem. Soc. 1992, 114, 75727574. (35) McIver, R. T., Jr.; Li, Y.; Hunter, R. L. Int. J. Mass Spectrom. Ion Processes 1994, 132, L1-7. (36) Yao, J.; Dey, M.; Pastor, S. J.; Wilkins, C. L. Anal. Chem. 1995, 67, 36383642. (37) Pasa-Tolic, L.; Huang, Y.; Guan, S.; Kim, H. S.; Marshall, A. G. J. Mass Spectrom. 1995, 30, 825-833. (38) Marshall, A. G. Adv. Mass Spectrom. 1989, 11A, 651-668. (39) Wanczek, K.-P. Int. J. Mass Spectrom. Ion Processes 1989, 95, 1-38. (40) Gord, J. R.; Freiser, B. S. Anal. Chim. Acta 1989, 225, 11-24. (41) Laude, D. A., Jr.; Hogan, J. D. Technisches Messen 1990, 57, 155-159. (42) Applications of Laser Desorption FTMS to Polymer and Surface Science; Cody, R. B., Jr., Bjarnason, A., Weil, D. A., Eds.; Oxford Univ. Press: New York, 1990; pp 316-339. (43) Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, 215A-229A. (44) Asamoto, B.; Dunbar, R. C. Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry; VCH: New York, 1991. (45) Campana, J. E. Proc. SPIE-Appl. Spectrosc. Mater. Sci. 1991, 138-149. (46) Marshall, A. G.; Schweikhard, L. Int. J. Mass Spectrom. Ion Processes 1992, 118/119, 37-70. (47) Ko ¨ster, C.; Kahr, M. S.; Castoro, J. A.; Wilkins, C. L. Mass Spectrom. Rev. 1992, 11, 495-512. (48) FTMS: Features, Principles, Capabilities, and Limitations; Jacoby, C. B., Holliman, C. L., Gross, M. L., Eds.; Kluwer Academic Publishers: Dordrecht, 1992; pp 93-116. (49) Pastor, S. J.; Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 379-384. (50) Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M., III; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1993, 4, 557-565. (51) Williams, E. R.; Henry, K. D.; McLafferty, F. W. J. Am. Chem. Soc. 1990, 112, 6157-6162. (52) Solouki, T.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Anal. Chem. 1995, 67, 4139-4144. (53) Guan, S.; Kim, H. S.; Marshall, A. G.; Wahl, M. C.; Wood, T. D.; Xiang, X. Chem. Rev. 1994, 94, 2161-2182. (54) Speir, J. P.; Gorman, G. S.; Pitsenberger, C. C.; Turner, C. A.; Wang, P. P.; Amster, I. J. Anal. Chem. 1993, 65, 1746-1752. (55) Guan, Z.; Drader, J. J.; Campbell, V. L.; Laude, D. A. Anal. Chem. 1995, 67, 1453-1458. (56) Strobel, F. H.; Solouki, T.; White, M. A.; Russell, D. H. J. Am. Soc. Mass Spectrom. 1991, 2, 91-94. (57) Jespersen, S.; Niessen, W. M. A.; Tjaden, U. R.; van der Greef, J.; Litborn, E.; Lindberg, U.; Roeraade, J. Rapid Commun. Mass Spectrom. 1994, 8, 581-584. (58) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem. 1993, 65, 574A-584A.

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tion of parent ions is particularly simple and effective with an FTICR instrument, in which sequential stages of an MSn experiment are separated in time, in contrast to beam mass analyzers, in which the stages are separated in space. Collisionally induced dissociation (CID) is probably the most explored among a variety of dissociation processes, e.g., electron impact dissociation (EI), surface-induced dissociation (SID), and photodissociation. Although several excitation techniques for multistage CID FT-ICR MSn experiments, including traditional resonant excitation, multiple excitation collisional activation (MECA),64 sustained offresonance irradiation (SORI),65,66 and very low energy (VLE) collisional activation,67,68 have been developed during the last decade, SORI is gaining primacy for CID FT-ICR mass analysis of biomolecules. In this method, ions excited by irradiation with off-resonance electric field pulse are slowly collisionally activated by repeated inelastic collisions, so that only the lowest energy pathways for dissociation are observed. In this paper, we show how the remeasurement technique may be used to recover product ions after each CID stage of a series of MSn experiments. Thus, it is not necessary to generate a new batch of ions to perform each stage of MSn, and less sample is required. Specifically, we present direct MALDI FT-ICR mass analyses of crude enzymatic digest mixtures of bovine cytochrome c and horse cytochrome c. High-resolution FT-ICR mass spectra of relatively impure samples provide rapid, sensitive, and reliable peptide mapping. Extraction of additional partial sequence information is illustrated by MSn experiments based on CID (by SORI) of tryptic fragment ions. In this way, peptides may be identified and partially sequenced from subfemtomole quantities. EXPERIMENTAL SECTION Equipment. MALDI FT-ICR mass spectra were acquired with an FTMS-2000 Fourier transform ion cyclotron resonance mass spectrometer (FTMS, Madison, WI), equipped with a 3 T superconducting magnet, dual cubic Penning traps, and an Odyssey data system. Laser desorption/ionization was performed with a cartridge-type pulsed N2 laser (Laser Science, Inc., Model VSL33ND, Newton, MA), operated at a wavelength of 337.1 nm, with a pulse width of 3 ns (laser power density, ∼106 W cm-2). The UV laser beam was directed through a quartz window on the analyzer side of the main vacuum chamber and focused by a 1 m focal length lens through a 2 mm diameter conductance limit to a spot size of ∼200 µm × 200 µm on the probe tip. Sample Preparation. Protein samples (horse cytochrome c, bovine cytochrome c, and trypsin) were purchased from Sigma (59) Han, X.; Gross, R. W. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10635-10639. (60) Anderen, P. E.; Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605-613. (61) Cody, R. B.; Tamura, J.; Finch, J. W.; Musselman, B. D. J. Am. Soc. Mass Spectrom. 1994, 5, 194-200. (62) Kelleher, N. L.; Senko, M. W.; Little, D. P.; O’Connor, P. B.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1995, 6, 220-221. (63) Valeskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aaseruud, D. J.; MacLafferty, F. W. Anal. Chem. 1995, 67, 3802-3805. (64) Lee, S. A.; Jiao, C. Q.; Huang, Y.; Freiser, B. S. Rapid Commun. Mass Spectrom. 1993, 7, 819-821. (65) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (66) Heck, A. J. R.; de Koning, L. J.; Pinkse, F. A.; Nibbering, N. M. M. Rapid Commun. Mass Spectrom. 1991, 5, 406-414. (67) Boering, K. A.; Rolfe, J.; Brauman, J. I. Int. J. Mass Spectrom. Ion Processes 1992, 117, 357-386. (68) Boering, K. A.; Rolfe, J.; Brauman, J. I. Rapid Commun. Mass Spectrom. 1992, 6, 303-305.

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Chemical Co. (St. Louis, MO) and used without further purification. Bovine cytochrome c and horse cytochrome c were dissolved in Tris buffer and digested with trypsin (1:50 by weight) at pH 8.2 and 37 °C. Digest mixtures were crudely purified with SepPak C18 miniextraction cartridges (Waters Chromatography Division, Millipore Corp., Milford, MA). 2,5-Dihydroxybenzoic acid (DHB) and D-fructose (Aldrich Chem. Co.) served as the matrix and comatrix, respectively, for all reported MALDI experiments. A 1 M stock solution of DHB matrix was prepared fresh daily in methanol acidified with 0.1% (v/v) trifluoroacetic acid. MALDI FT-ICR mass spectra were obtained at a typical matrix:comatrix:analyte ratio of ∼1000:500: 1. Approximately 10 µL of the solution mixture containing analyte and matrix was applied to the solids insertion probe tip and allowed to dry in air before insertion into the mass spectrometer. FT-ICR Experimental Event Sequence. High-resolution MALDI FT-ICR mass spectra were acquired in the analyzer compartment of a dual cubic ion trap. All MSn experiments were carried out in the source compartment. High-Resolution Mass Spectra. After the laser desorption/ ionization event, the ion z-axis translational energy was minimized by use of gated deceleration at (9.75 V. Thus, for positive-ion trapping, the sample probe, source trap plate, and conductance limit plate during the laser desorption event (60-150 µs) were biased to 0.0, 0.0, and +9.75 V, respectively.69 After 60-150 µs to allow for ion transfer into the trap, the source trap and conductance limit plates were each restored to +2 V. Axialization was provided by a train of identical low-amplitude SWIFT excitations, in azimuthal quadrupolar mode, at the cyclotron frequency of the ion(s) of interest in the presence of the collision gas (Ar at partial pressure of ∼2 × 10-7 in the source trap, corresponding to ∼2 × 10-9 Torr in the analyzer trap). A Varian leak valve (Varian Associates, Walnut Creek, CA) was used to maintain a constant neutral background pressure in the ion trap mass analyzer. Following ion transfer to the low-pressure analyzer trap, the electrostatic trapping potential was lowered adiabatically to e(0.5 V. Conventional frequency sweep excitation from 1 to 500 kHz at a 500 Hz/µs sweep rate was followed by heterodyne detection to yield 256K time domain data points. Each FT-ICR mass spectrum resulted from a single laser shot, based on direct FFT (and magnitude calculation) of the unapodized time domain data, unless stated otherwise. MSn Experiments. Our experimental event sequence for successive MSn experiments is shown in Figure 1. MALDIgenerated ions of initially high kinetic energy are decelerated as described above. The trapped ions are allowed to relax axially to the center of the trap for several seconds in the presence of argon (∼2 × 10-7 Torr). Ion cyclotron motion is then excited by frequency sweep irradiation, for which the sweep rate and radio frequency amplitude are optimized for each sample. Typically, coherent ICR motion is excited by dipolar frequency sweep excitation (∼132 Vp-p amplitude, 1-500 kHz at a sweep rate of 700 Hz/µs). Fourier transformation of the resulting discrete time domain signal (32K data, 1 MHz Nyquist bandwidth), without zerofilling or apodization, followed by magnitude calculation and frequency-to-mass conversion, yields an FT-ICR parent ion mass spectrum (e.g., Figure 4A). (69) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993, 65, 2621-2627.

Figure 1. Experimental event sequence for multiple remeasurement of MS, MS2, and MS3 from a single ionization event. First, parent ions over the full m/z range are excited and detected. Selected parent ions are then isolated by radial ejection of ions of undesired m/z values by stored waveform inverse Fourier transform (SWIFT) broadband excitation. Parent ion selection is confirmed by excitation and detection of the m/z-selected ions. Parent ion dissociation is accomplished by sustained off-resonance irradiation (SORI) at ∼800 Hz below the reduced ion cyclotron frequency of the parent ion, and the daughter ions over the m/z range of interest are then axialized by n ) 15 repeated broadband azimuthal quadrupolar excitation events. Daughter ions are then excited and detected. The process is then repeated to select and dissociate daughter ions of a particular m/z ratio, followed by excitation and detection to yield a granddaughter ion mass spectrum. In the final MS stage, ions are axialized x ) 15 times after each of y ) 15 remeasurements.

Stored-waveform inverse Fourier transform (SWIFT)70 radial ejection then removes parent ions of all but a selected m/z ratio(s), and the mass-selected parent ions are (re)detected following chirp excitation (e.g., Figure 4B). The mass-selected parent ions are then translationally excited to dissociate by means of collisional activation provided by sustained off-resonance irradiation (SORI) at ∼800 Hz below the reduced ion cyclotron frequency of the parent ion. Azimuthal quadrupolar excitation, consisting of a series of 15 SWIFT waveforms (see Figure 1) irradiating the mass range of interest serves to axialize the product (“daughter”) ions, which are rendered observable by chirp excitation and detection and Fourier transformed without apodization or zero-filling to yield an MS2 magnitude mode mass spectrum (e.g., Figure 4C). (A home-built relay circuit switches between dipolar excitation and detection modes to azimuthal quadrupolar axialization mode.54,71) After another SWIFT ejection event to isolate mass-selected daughter ions, an additional SORI excitation serves to dissociate those daughter ions, and the resulting granddaughter ions are axialized repeatedly (x times in Figure 1) and remeasured repeatedly (y times in Figure 1). The time domain signal from y ) 15 summed acquisitions, in which each acquisition included x ) 15 axialization cycles (each containing 15 repeated waveforms), are Fourier transformed without apodization or zero filling to yield the MS3 magnitude mode granddaughter ion mass spectrum (e.g., Figure 4D). RESULTS AND DISCUSSION Here we report MALDI FT-ICR mass spectral analysis of relatively impure tryptic digest mixtures of horse cytochrome c (70) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (71) Guan, S.; Marshall, A. G.; Wahl, M. Anal. Chem. 1994, 66, 1363-1367.

Figure 2. MALDI FT-ICR positive-ion high-resolution mass spectrum of a bovine cytochrome c tryptic digest solution.

and bovine cytochrome c. The pancreatic proteolytic enzyme, trypsin, attacks peptide bonds specifically at the carboxyl end of either lysine or arginine, and MALDI mass spectrometric mapping of tryptic fragments gives a fingerprint of the corresponding protein. Variants of cytochrome c, an electron-transfer protein bound to the mitochondrial membrane and containing a heme prosthetic group covalently attached to two cysteine side chains, typically exhibit high sequence homology and consequently similar conformations in solution.72 Since these proteins are involved in the respiratory chain of all aerobic organisms, primary structures of most variants are known; three-dimensional structures of some variants have been obtained by use of x-ray diffraction; and tertiary structures in solution have been investigated by circular dichroism and NMR techniques.73-75 Capillary electrophoresis/mass spectrometry peptide maps of horse, bovine, and dog cytochrome c are available.76 Mass spectrometry easily and clearly detects differences in primary structure between peptides differing by only three (out of 104) amino acid residues. More recently, ESI FTICR MSn (n e 4) experiments on different cytochrome c variants have differentiated subtle differences between their primary structures.10 Here we report complementary high-resolution MALDI FT-ICR mass spectra of horse and bovine cytochrome c tryptic digest mixtures. A (somewhat low) total coverage of ∼75% of the primary sequence was obtained in both cases (see below), due to incomplete enzymatic digestion and to the choice of MALDI experimental parameters (i.e., long deceleration period, chosen to suppress signals corresponding to low-mass ions). MSn of selected tryptic fragments for several stages of low-energy SORICID from a single laser shot are presented. FT-ICR MS, MS2, and MS3 spectra have been obtained from ∼1 pmol of peptide. MALDI FT-ICR Positive-Ion Mass Spectrum from a Bovine Cytochrome c Tryptic Digest Solution. Figure 2 shows a portion of the high-resolution analyzer side positive-ion MALDI (72) Stryer, L. Biochemistry; W. H. Freeman and Co.: New York, 1988. (73) Takano, T.; Kallai, O. B.; Swanson, R.; Dickerson, R. E. J. Biol. Chem. 1973, 218, 5234-5255. (74) Goto, Y.; Takahashi, N.; Fink, A. I. Biochemistry 1990, 29, 3480-3488. (75) Hawkins, B. K.; Hilgen-Willis, S.; Pielak, G. J.; Dawson, J. H. J. Am. Chem. Soc. 1994, 116, 3111-3112. (76) Takada, Y.; Nakayama, K.; Yoshida, M.; Sakairi, M. Rapid Commun. Mass Spectrom. 1994, 8, 695-697.

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Figure 3. MALDI FT-ICR positive-ion high-resolution mass spectrum of a horse cytochrome c tryptic digest solution. Fragments observed here are either the same as or complementary to those from the negative-ion mass spectrum (not shown) and from MS2 and MS3 experiments (Figures 4 and 5). Comparison of the experimentally observed ion abundances with theoretically calculated isotopic distributions reveals relative contributions from loss of H2O and NH3 neutrals from tryptic fragment ions.

FT-ICR magnitude mode mass spectrum of tryptic fragments of bovine cytochrome c. The inset expands the 2009 < m/z < 2013 region corresponding to (GITWGEETLMEYLENPK + H)+ tryptic digest fragment ions (residues 56-72) at m/z 2009.95, specific to bovine cytochrome c. Note that FT-ICR MS resolves the 13C isotopic distribution of the (GITWGEETLMEYLENPK + H)+ enzymatic digest fragment, which matches the calculated isotopic relative abundance for this fragment ion within ∼5%. These ions are also observed in a negative-ion MALDI FT-ICR mass spectrum from the bovine cytochrome c tryptic digest (not shown). Signals arising from residues 56-72 are not seen in the MALDI FT-ICR mass spectra of horse cytochrome c. 60Gly in bovine cytochrome c is replaced by 60Lys in horse cytochrome c. Thus, the bovine cytochrome c tryptic digest fragment, GITWGEETLMEYLENPK, is replaced by GITWK (m/z ≈ 604.35) and EETLMEYLENPK (m/z ≈ 1495.70) fragments in horse cytochrome c tryptic digest mixture. MALDI FT-ICR Positive-Ion Mass Spectrum from a Horse Cytochrome c Tryptic Digest Solution. Figure 3 shows a MALDI FT-ICR positive-ion mass spectrum of tryptic fragments of horse cytochrome c. Insets show m/z-expanded regions near m/z 1169 and 1616, corresponding to (TGPNLHGLFGR + H)+ (residues 28-38) and ([IFVQKCAQCHTVEK + H] - H2O/NH3)+ (residues 9-22); both fragments are common to horse and bovine cytochrome c. Although the observed 13C isotope pattern for the m/z 1169 fragment again closely matches the calculated distribution, the isotopic distribution near m/z ≈ 1616 represents a sum of two isotopic distributions corresponding to the loss of different neutrals (H2O or NH3). Relatively large variations (>30%) in the observed relative isotope abundances (not shown here) for ([IFVQKCAQCHTVEK + H] - H2O/NH3)+ ions from one laser shot to another are presumed to originate from variations in parent ion internal energy, which in turn determines the predominance of H2O or NH3 loss. We compared the experimentally observed 3722 Analytical Chemistry, Vol. 68, No. 21, November 1, 1996

Figure 4. MALDI FT-ICR mass spectra of horse cytochrome c tryptic digest. From the parent ion mass spectrum (A), ions of m/z 1616 are isolated (B) and collisionally dissociated to yield the MS2 spectrum (C), from which ions of m/z 1487 are isolated and collisionally dissociated to yield an MS3 spectrum (D). The filled circles (b) denote the ions observed at each stage of ion isolation and/or ion dissociation.

ion abundance with theoretically calculated isotopic ratios to estimated the relative loss of H2O and/or NH3 from tryptic fragment ions. The observed pattern for isotopic distribution of ions near m/z ≈ 1616 indicates that during and/or after the laser desorption process, parent ions near m/z ≈ 1633.8 lose small neutrals (in this case, ∼91% H2O and ∼9% NH3). The examples in Figures 2 and 3 illustrate the great value of the high mass resolving power routinely obtainable in MALDI FT-ICR MS for assignment of fragment ions in mass spectra of enzymatic digest mixtures of proteins. At typical FT-ICR mass resolving power, m/∆m50% ≈ 100 000 at m/z ≈ 1000-2000 at 3 T, 13C isotopes are clearly resolved, and the ion relative abundances may be compared with calculated isotopic distributions to establish correct neutral loss assignments. By capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI MS), Takada et al. observed cytochrome c tryptic digest product ions at m/z 1635, assigned as (CAQCHTVEK + heme)+.76 From our MALDI FT-ICR results (Figures 3 and 4), we are able to assign the ions at m/z 1634 to the [IFVQKCAQCHTVEK + H]+ fragment. Apparently, upon fragmentation of the heme group, cysteine residues are reduced as a result of the MALDI process. Although the mechanism for this reaction is not well understood, similar rearrangement/reduction processes from photofragmentation of metal-containing porphyrins have previously been reported.77 In addition, in both negative(not shown) and positive-ion modes, we observe ions of m/z ≈ 1018, corresponding to CAQCHTVEK fragments produced by additional digestion of IFVQKCAQCHTVEK. Moreover, the 13C isotopic distribution for ions of m/z ≈ 1616, as well as CID MSn spectra (see Figures 3 and 4), supports the proposed (IFVQKCAQCHTVEK + H]+ assignment. ESI is evidently a “softer” ionization technique, and the heme group remains intact during the ionization process. Single Laser Shot MALDI FT-ICR MS, MS2, and MS3 Mass Spectra. A primary aim of this study is to develop a reliable (77) Solouki, T.; Russell, D. H. Appl. Spectrosc. 1993, 47, 211-217.

Figure 5. MALDI FT-ICR positive-ion mass spectra of horse cytochrome c tryptic digest. Selected ions of m/z 1169 are isolated, and MS, MS2, and MS3 mass spectra from the same batch of ions are obtained. The circles denote the observed ions at each stage of multiple ion remeasurement. The filled circles (b) denote the isolated ions at each stage of ion isolation and/or ion dissociation. Comparison of the experimentally observed ion abundances with theoretically calculated isotopic distributions reveals the relative contributions from loss of H2O and NH3 neutrals from tryptic fragment ions.

method for peptide/protein characterization on a subpicomole level. The sensitivity of MALDI FT-ICR MS analysis can be improved drastically by careful sample preparation combined with the remeasurement technique.52 However, tandem mass spectrometry is required to yield sequence information. Traditionally, each stage of a multistage MSn experiment consumes at least a few femtomoles of sample. Fortunately, nondestructive FT-ICR detection allows acquisition of multistage MSn (n ) 1, 2, 3) spectra from the same batch of ions initially formed by laser desorption/ ionization. Figures 4 and 5 illustrate this approach, not available from scanning mass spectrometers, showing MSn (n ) 1, 2, 3) spectra of two horse cytochrome c tryptic digest fragment ions. A relatively large number of parent ions can be generated and trapped by optimizing the matrix-to-analyte ratio and the deceleration potential69 that reduces parent ion kinetic energy and thereby increases trapping efficiency. Relaxation of the trapped parent ions in the presence of 2 × 10-7 Torr of Ar in the source trap was followed by standard excitation and detection events to yield the spectrum in Figure 4A. After detecting the primary ion mass spectrum, we damped ion cyclotron motion by collisions with background neutrals (argon static pressure of 2 × 10-7 Torr). Ions of a single isotopic m/z cluster were isolated by SWIFT broadband radial ejection, and a second detection event served to confirm isolation of parent ions of m/z ≈ 1616 (Figure 4B). This stage corresponds to the middle segment of a triple-quadrupole instrument, with the advantage that we are able to monitor (as well as isolate) parent ions in that stage. CID of SWIFT-isolated parent ions was then performed by offresonance (SORI) excitation, followed by broadband axialization for ∼15 s to return the daughter ions near to the trap central axis. Although our SORI and axialization events were conducted sequentially, rather than simultaneously as in prior experiments,9,71

daughter ions were nevertheless recovered at high efficiency, presumably because of the relatively high mass of the daughter ions and relatively low pressure (leading to relatively slow outward radial diffusion of ions due to magnetron radius expansion by ionneutral collisions). The primary CID fragments from the parent ions (TGPNLHGLFGR + H)+ at m/z ≈ 1169 (see Figure 5) and ([IFVQKCAQCHTVEK + H] - H2O/NH3)+ (see Figure 4) at m/z ≈ 1616 are ([TGPNLHGLFGR + H] - H2O/NH3)+ and ([IFVQKCAQCHTVEK + H] - K)+, as shown in the MS2 mass spectra of Figures 4C and 5 (middle), (corresponding to the third segment of a triple-quadrupole instrument). After relaxation, selected daughter ions of m/z ≈ 1487 (in Figure 4) and m/z ≈ 1151 (in Figure 5) were SWIFT-isolated and dissociated in a second SORI-CID event. The axializationexcitation-detection sequence, i.e., remeasurement, was repeated 15 times, and the summed time domain signals were processed to give the MS3 mass spectra shown at the bottom of Figures 4 and 5. The most abundant secondary product ions are ([TGPNLHGLFGR + H] - H2O/NH3 - R)+ and ([IFVQKCAQCHTVEK + H] - K - E)+. Obtaining just those two spectra would require a pentaquadrupole instrument if performed tandem-in-space rather than tandem-in-time. Note that, at higher collision gas pressure (>10-6 Torr), we are able to observe several fragment ions in a single experiment; however, to observe sequential fragmentation of a specific parent ion from a single laser shot, we intentionally chose conditions that produce few fragment ions (see discussion of CID efficiency). Choice of Experimental Conditions. The present MSn spectra were obtained by use of SORI pulses of 15.75 Vp-p amplitude, at ∼800 Hz below the observed ICR frequency of the parent ions. Under those conditions, the average center-of-mass translational energies of the (TGPNLHGLFGR + H)+ and (IFVQKCAQCHTVEK + H)+ ions were 0.49 and 0.26 eV. Collisional damping rate constants, computed from a hard-sphere model for ion-neutral collisions,78 are 0.3 and 0.2 s-1, corresponding to less than 10 ion-neutral collisions during the SORI event. Generally, the extent of dissociation may be controlled by varying the pressure, the difference between SORI and ICR frequencies, and the amplitude and duration of SORI excitation. Here, we required SORI excitation frequency close to the ICR frequency and lengthy SORI excitation duration (g10 s), to induce dissociation (e.g., (IFVQKCAQCHTVEK + H)+) or even dehydration (e.g., (TGPNLHGLFGR + H)+), due to the low collision frequency,