Anal. Chem. 2002, 74, 3877-3886
Multiple Ion Isolation Applications in FT-ICR MS: Exact-Mass MSn Internal Calibration and Purification/Interrogation of Protein-Drug Complexes Gary Kruppa,†,§ Paul D. Schnier,‡ Keiko Tabei,‡ Steven Van Orden,† and Marshall M. Siegel*,‡
Bruker Daltonics Inc., Billerica, Massachusetts 01821, and Wyeth Research, Pearl River, New York 10965
Two new applications using multiple ion isolations in the cell of a Fourier transform-ion cyclotron resonance mass spectrometer equipped with an electrospray ionization source are described. A procedure that uses multiple ion isolations of an analyte and calibrants for internal calibration at each stage in a MSn experiment, under highresolution exact-mass conditions, for structural characterization/elucidation of angiotensin I and rapamycin is illustrated. Fragment ion mass accuracies 100 ppm. Low-parts-per-million mass accuracies can be achieved if the ion abundances of the analytes are maintained at levels approximately equal to that of the calibrants. Internal calibration corrects for this major cause of mass measurement error in FTMS, since all ions experience a similar effect, leading to sub-parts-per-million mass accuracies. For structural elucidation work with unknowns, the use of internal calibration is the best method for achieving the level of accuracy needed to obtain an elemental formula. It is difficult to directly obtain a unique elemental formula for unknowns with masses above 500 Da simply from an exact-mass measurement. This can be remedied by use of the elemental formulas computed from the exact-mass measurements of MS2 fragment ions with masses below 500 Da. For MS2 experiments performed with tandem mass spectrometers, such as magnetic sector and triple quadrupole instruments, only a single mass or mass range can be selected by the first mass spectrometer and transmitted to the collision cell. Ion (5) Walk, T. B.; Trautwein, A. W.; Bandel, H.; Jung, G. Comb. Chem. 1999, 561-587. (6) Schmid, D. G.; Grosche, P.; Bandel, H.; Jung, G. Biotechnol. Bioeng. 2001, Volume Date 2000-2001, 71 (2), 149-161. (7) He, H.; Shen, B.; Korshalla, J.; Siegel, M. M.; Carter, G. T. J. Antibiot. 2000, 53 (2), 191-195. (8) Shi, S. D.-H.; Hendrickson, C. L.; Marshall, A. G.; Siegel, M. M.; Kong, F.; Carter, G. T. J. Am. Soc. Mass Spectrom. 1999, 10 (12), 1285-1290. (9) Huang, N.; Siegel, M. M.; Kruppa, G. H.; Laukien, F. H. J. Am. Soc. Mass Spectrom. 1999, 10 (11), 1166-1173. (10) Shen, Y.; Tolic, N.; Zhao, R.; Pasa-Tolic, L.; Li, L.; Berger, S. J.; Harkewicz, R.; Anderson, G. A.; Belov, M. E.; Smith, R. D. Anal. Chem. 2001, 73 (13), 3011-3021. (11) Pasa-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 (34), 7949-7950. (12) Hofstadler, S. A.; Griffey, R. H. Curr. Opin. Drug Discovery Dev. 2000, 3 (4), 423-431. (13) Griffey, R. H.; Sannes-Lowery, K. A.; Drader, J. J.; Mohan, V.; Swayze, E. E.; Hofstadler, S. A. J. Am. Chem. Soc. 2000, 122 (41), 9933-9938. (14) Griffey, R. H.; Hofstadler, S. A.; Sannes-Lowery, K. A.; Ecker, D. J.; Crooke, S. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (18), 10129-10133. (15) Schnier, P. D.; McDonald, L. A.; Siegel, M. M. Proceedings of the American Society for Mass Spectrometry and Allied Topics, 48th ASMS Conference, Long Beach, CA, June 11-16, 2000; pp 695-696. (16) Winger, B. E.; Kemp, C. A. J. Am. Pharm. Rev. 2001, 4 (2), 55-56, 58, 60, 62-63. (17) Easterling, M. L.; Mize, T. H.; Amster, I. J. Anal. Chem. 1999, 71 (3), 624632.
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trapping mass spectrometers, including FTMS instruments, are unique in that a variety of ions of different masses or mass ranges may be isolated, and individual ions or mass ranges may be selected for activation and fragmentation. A unique FTMS application for multiple ion isolations is that several calibrant ions may be isolated along with the desired parent ion, and only the parent ion may be activated to produce a product ion spectrum, all performed under exact-mass high-resolution conditions. The spectrum can be internally calibrated using the reference masses of the unactivated calibrant peaks that remain trapped in the analyzer cell. This procedure can be repeated for the calibrant ions and selected parent ions at each stage of the MSn experiments to produce internally calibrated MSn spectra. Some previous reports have shown that mixtures of ions produced by a variety of methods can be used to internally calibrate MS2 spectra in FTMS to obtain low parts-per-million mass accuracies of fragment ions.18-20 Tutko, et al.18 used multiple ionization techniques in which the analyte was ionized by UV-MALDI while the reference ions were produced independently by EI and together mixed in the FTMS cell. Hannis and Muddiman19 utilized two independent electrospray nozzles to alternately deliver analyte and reference ions into an accumulation hexapole prior to transfer into a FTMS cell. Hofstadler, et al.20 used IRMPD to fragment relatively unstable oligonucleotides in the presence of proteins and peptides of high thermal stability to internally calibrate FTMS IRMPD spectra. These examples did not involve the use of multiple ion isolations. In this work, we demonstrate that the use of multiple ion isolations at each stage of MSn leads to a cleaner and easier to interpret MSn spectra. Another application of multiple ion isolations prior to MSn unique for ion trapping mass analyzers is the enhancement of the signal-to-noise ratio in MSn spectra of multiply charged ions. In ESI-MS spectra of mixtures of proteins or peptides, distributions of charge states are normally observed. The selection of a single charge state for collisional activation in a tandem mass spectrometer effectively discards the ion current contained in the other charge states of the same molecule. With ion trapping instruments, the entire charge state distribution of an individual protein or peptide can be cleanly isolated from the other components in the mixture. Fragmentation of all of the charge states simultaneously can be achieved with multiple RF excitation of all the charge states by sustained off-resonance irradiation with collision-induced dissociation (SORI-CID)21 in the presence of a gas or by IR-laser multi-photon dissociation (IRMPD)22-27 in the absence of a gas. The signal-to-noise ratios of the fragment ions generated from the (18) Tutko, D. C.; Henry, K. D.; Winger, B. E.; Stout, H.; Hemling, M. Rapid Commun. Mass Spectrom. 1998, 12 (6), 335-338. (19) Hannis, J. C.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2000, 11 (10), 876-883. (20) Hofstadler, S. A.; Griffey, R. H.; Pasa-Tolic, L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1998, 12 (19), 1400-1404. (21) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246 (1), 211-225. (22) Hunt, D. F.; Shabanowitz, J.; Yates, J. R., III. J. Chem. Soc., Chem. Commun. 1987, (8), 548-550. (23) Hunt, D. F.; Shabanowitz, J.; Yates, J. R., III; Griffin, P. R.; Zhu, N. Z. Anal. Chim. Acta 1989, 225 (1), 1-10. (24) Griffin, P. R.; Shabanowitz, J.; Yates, J. R., III; Zhu, N. Z.; Hunt, D. F. Technol. Protein Chem. 1989, 160-167. (25) Shabanowitz, J.; Hunt, D. F. Oxford Ser. Opt. Sci. 1990, 1, 340-352. (26) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66 (18), 2809-2815.
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multiply charged ions are considerably enhanced versus the case in which only a single charge state is isolated and fragmented. The experimental isolation of multiple charge states of an individual component in the presence of a mixture corresponds to purification and concentration of the component of interest. To further maximize the information content of the samples, FTMSn experiments can be performed under high-resolution exact-mass conditions to obtain the elemental formulas of the fragmentation products. Two experimental techniques for multiple ion isolations have been developed that are capable of notching out frequency (mass) regions for isolating the desired components and are referred to as stored waveform inverse Fourier transformation (SWIFT),28-32 and correlated harmonic excitation fields (CHEF).33-38 Nearly all of the previously reported FTMS mass spectral ion isolation applications have involved the isolation of only a single component during each step in the MSn analysis; however, a few illustrations of multiple ion isolations have been reported. The earliest reports of multiple ion isolations were used with quadrupole ion-trap mass spectrometers. Kenny et al.39 isolated two different compounds from a complex mixture, and a MS2 spectrum of each component was obtained. In addition, Soni and Cooks40 demonstrated the acquisition of MS spectra after isolation of two related ions of L-carnatine and the ability to isolate a peptide in the presence of a number of cesium iodide ion adducts for internal calibration. More recently, using FTMS instrumentation, Smith and co-workers demonstrated the ability to selectively enhance low-abundance ions after suppressing selectively and simultaneously a number of abundant ions41 and the ability to select simultaneously a number of peptide ions both manually and with data-dependent scanning for multiplexed MS2 experiments using IRMPD and SORI-CID methods.42,43 Also recently, Wu illustrated “multistage accurate mass spectrometry” by selecting an analyte and reference ions using SWIFT for calibration of MS2, (27) Little, D. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1996, 7 (2), 209-210. (28) Wang, T. C. L.; Ricca, T. L.; Marshall, A. G. Anal. Chem. 1986, 58 (14), 2935-2938. (29) Chen, L.; Wang, T. C. L.; Ricca, T. L.; Marshall, A. G. Anal. Chem. 1987, 59 (3), 449-454. (30) Chen, L.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1987, 79 (1), 115-125. (31) Guan, S.; Marshall, A. G. Anal. Chem. 1993, 65 (9), 1288-1294. (32) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 5-37. (33) Heck, A. J. R.; De Koning, L. J.; Pinkse, F. A.; Nibbering, N. M. M. Rapid Commun. Mass Spectrom. 1991, 5 (9), 406-414. (34) de Koning, L. J.; Nibbering, N. M. M.; van Orden, S. L.; Laukien, F. H. Int. J. Mass Spectrom. Ion Processes 1997, 165/166, 209-219. (35) Heck, A. J. R.; Derrick, P. J. Anal. Chem. 1997, 69 (17), 3603-3607. (36) Heck, A. J. R.; O’Sullivan, M. L.; Derrick, P. J. Adv. Mass Spectrom. 1998, 14, C026140/1-C026140/6. (37) Heck, A. J. R.; Derrick, P. J. Eur. Mass Spectrom. 1998, 4 (3), 181-188. (38) Kawashima, K. J. Mass Spectrom. Soc. Jpn. 1999, 47 (3), 160-163. (39) Kenny, D. V.; Gallahan, P. J.; Gordon, S. M. Rapid Commun. Mass Spectrom. 1993, 7 (12), 1086-1089. (40) Soni, M. H.; Cooks, R. G. Anal. Chem. 1994, 66 (15), 2488-2496. (41) Bruce, J. E.; Anderson, G. A.; Smith, R. D. Anal. Chem. 1996, 68 (3), 534541. (42) Masselon, C.; Anderson, G. A.; Harkewicz, R.; Bruce, J. E.; Pasa-Tolic, L.; Smith, R. D. Anal. Chem. 2000, 72 (8), 1918-1924. (43) Li, L.; Masselon, C. D.; Anderson, G. A.; Pasa-Tolic, L.; Lee, S.-W.; Shen, Y.; Zhao, R.; Lipton, M. S.; Conrads, T. P.; Tolic, N.; Smith, R. D. Anal. Chem. 2001, 73 (14), 3312-3322.
whereas for stages greater than MS2, no additional isolations were utilized.44 In this report, the CHEF technique, developed originally for the isolation of single ions, is extended for the isolation of a multiple number of ions by notching out multiple frequencies (masses). We refer to this technique as multi-CHEF.45,46 Here, the multi-CHEF technique is used to isolate multiple ions in exactmass internal calibration experiments at each stage of MSn. Additionally, multi-CHEF is used to isolate multiple charge states of protein-drug complexes in the presence of unreacted protein for purification, concentration, fragmentation, and structural elucidation of the protein-drug complex. EXPERIMENTAL SECTION Bruker-Daltonics Apex II FT mass spectrometers were used for these studies. They were equipped with 4.7 or 9.4 T superconducting magnets, an Analytica electrospray source, and a Synrad 50 W IRMPD laser. All small molecules used were commercially available or synthesized in-house. The epidermal growth factor receptor (EGFr) protein (Mw 62.2 kDa) was prepared in-house using standard recombinant technology. Reference compounds adenosine monophosphate (AMP, C10H14N5O7P, Mw 347.063 09), bradykinin (C50H73N15O11, Mw 1059.561 40), and melittin [C131H229N39O31, Mw 2844.754 17] were used to calibrate the instrument for MS2 studies of angiotensin I (C62H89N17O14, Mw 1295.677 49). The Agilent ESI-MS tuning mixture (ES Tuning Mix, PN G2421A) with reference masses at m/z 622.028 95, 922.009 79, and 1521.971 46 were used to calibrate the instrument for MS3 studies of rapamycin (C51H79NO13, Mw 913.551 31). Typically, a minimum of three calibration points is needed to calibrate the mass ranges acquired for FTMSn spectra. The calibration procedure, using the Francl equation, has been described by Shi et al.47 Extrapolations to low mass are achieved routinely with highest accuracy, namely,
