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Automated Gain Control and Internal Calibration with External Ion Accumulation Capillary Liquid Chromatography-Electrospray Ionization-Fourier Transform Ion Cyclotron Resonance Mikhail E. Belov, Rui Zhang, Eric F. Strittmatter, David C. Prior, Keqi Tang, and Richard D. Smith*
Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352
When combined with capillary LC separations, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) has demonstrated capabilities for advanced characterization of proteomes based upon analyses of proteolytic digests. Incorporation of external (to the ICR cell) multipole devices with FTICR for ion selection and ion accumulation has enhanced the dynamic range, sensitivity, and duty cycle of measurements. However, the highly variable ion production rate from an LC separation can result in “overfilling” of the external trap during the elution of major peaks and result in m/z discrimination and fragmentation of peptide ions. Excessive space charge trapped in the ICR cell also causes significant shifts in the detected ion cyclotron frequencies, reducing the achievable mass measurement accuracy (MMA) and making protein identification less effective. To eliminate m/z discrimination in the external ion trap, further increase duty cycle, and improve MMA, we have developed the capability for datadependent adjustment of ion accumulation times in the course of an LC separation, referred to as automated gain control (AGC). This development has been implemented in combination with low kinetic energy gated ion trapping and internal calibration using a dual-channel electrodynamic ion funnel. The overall system was initially evaluated in the analysis of a tryptic digest of bovine serum albumin. In conjunction with internal calibration, the capillary LC-ESI-AGC-FTICR instrumentation provided a ∼10-fold increase in the number of identified tryptic peptides compared to that obtained using a fixed ion accumulation time and external calibration methods. Over the past few years, capillary liquid chromatography/mass spectrometry (LC/MS) has increasingly been employed in the analysis of the protein complement of a cell, tissue, or organism present under various physiological conditions (i.e., its proteomes).1-6 When coupled to LC separations,7 Fourier transform (1) Yates, J. R.; Carmack, E.; Hays, L.; Link, A. J.; Eng, J. K. Math. Mol. Biol. 1999, 112, 553-569. (2) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (3) Yates, J. R. Trends Genet. 2000, 17, 81-87. 10.1021/ac0206770 CCC: $25.00 Published on Web 07/08/2003
© 2003 American Chemical Society
ion cyclotron resonance (FTICR) mass spectrometry8,9 has been shown to provide ultrasensitive, high-mass-measurement-accuracy capabilities for characterization of proteolytic digests.10,13 The sensitivity, dynamic range, and duty cycle provided by FTICR has been shown to be increased by ion trapping and accumulation in a 2D rf-only multipole trap (e.g., linear octopole,14,15 hexapole,16 or quadrupole10) positioned externally to an FTICR mass spectrometer. However, the advantages of the external ion accumulation are substantially negated if ion accumulation is accompanied by a significant “bias” across the m/z range. We recently reported on two major mechanisms of ion discrimination that apply when accumulating large ion populations in an rf-only quadrupole.17 These include (1) the radial ion separation due to the m/zdependent balance of the effective potential force18 and space charge repulsion (i.e., radial stratification in an rf-only quadrupole), resulting in discrimination against higher m/z ions, and (2) the space charge-induced instability of lower m/z ions. In addition to such processes causing m/z discrimination, pronounced ion (4) Smith, R. D. Int. J. Mass Spectrom. 2000, 200, 509-544. (5) Godovac-Zimmerman, J.; Brown, L. Mass Spectrom. Rev. 2000, 20, 1-57. (6) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-295. (7) Stockton, G. W.; Meek, J. T.; Millen, W. G.; Wayne, R. S. In FT-ICR/MS: Analytical Applications of Fourier Transfrom Ion Cyclotron Resonance Mass Spetrometry; Asamoto, B., Ed.; VCH: New York, 1991; pp 235-272. (8) Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 25, 282. (9) Marshall, A. G. Int. J. Mass Spectrom. 2000, 200, 331-356. (10) Belov, M. E.; Nikolaev, E. N.; Anderson, G. A.; Udseth, H. R.; Conrads, T. P.; Veenstra, T. D.; Masselon, C. D.; Gorshkov, M. V.; Smith, R. D. Anal. Chem. 2001, 73, 253-261. (11) Shen, Y.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K.; Pasa-Tolic, L.; Veenstra, T. D.; Lipton, M. S.; Smith, R. D. Anal. Chem. 2001, 73, 1766-1775. (12) Conrads, T. P.; Alving, K.; Veenstra, T. D.; Belov, M. E.; Anderson, G. A.; Anderson, D. J.; Pasa-Tolic, L.; Chrisler, W. B.; Trall, B. D.; Smith, R. D. Anal. Chem. 2001, 73, 2132-2139. (13) Shen, Y.; Tolic, N.; Zao, R.; Pasa-Tolic, L.; Li, L.; Berger, S. J.; Harkewicz, R.; Anderson, G. A.; Belov, M. E.; Smith, R. D. Anal. Chem. 2001, 73, 30113021. (14) Senko, M. W.; Hendrikson, C. L.; Emmett, M. R.; Shi, S. D-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (15) Quenzer, T. L.; Emmett, M. R.; Hendrickson, C. L.; Kelly, P. H.; Marshall, A. G. Anal. Chem. 2001, 73, 1721-1725. (16) Sannes-Lowery, K.; Griffey, R. H.; Kruppa, G. H.; Speir, J. P.; Hofstadler, S. A. Rapid Commun. Mass Spectrom. 1998, 12, 1957-1961. (17) Belov, M. E.; Nikolaev, E. N.; Harkewicz, R.; Masselon, C. D.; Alving, K.; Smith, R. D. Int. J. Mass Spectrom. 2001, 208, 205-225. (18) Dehmelt, H. G. Adv. At. Mol. Phys. 1967, 3, 53.
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fragmentation has also been observed when higher space charge levels are trapped in an rf-only hexapole16,19,20 or quadrupole.21 The onset and degree of ion fragmentation in an rf-only quadrupole was shown to be a function of the ratio of the radial (i.e., the effective potential from an rf-field) and axial potential wells (i.e., the potential difference between the quadrupole rod bias and the potential applied to the conductance limits confining the quadrupole).21 Space charge accumulated in an rf-only multipole causes ion cloud expansion from the center of the accumulation multipole to larger radii (i.e., higher rf motion amplitudes), where ions gain additional energy from the rf-field and can decompose in collisions with background gas (multipole storage-assisted dissociation16). These problems are further exacerbated by the higher ion currents provided by the use of an electrodynamic ion funnel in the electrospray ionization(ESI)-FTICR interface22 and can considerably impair additional efforts to enhance the sensitivity of FTICR mass spectrometer measurements. We recently demonstrated that bias effects in the accumulation of larger ion populations in a 2D rf-only quadrupole can be mitigated using either lower amplitude auxiliary dipolar excitation superimposed over the main rf field23 or a pulsed increase in the ambient gas pressure in the external trap during the accumulation periods.24 These approaches resulted in the disruption of ion discrimination processes at longer accumulation periods in the rf-only quadrupole. However, quantitative LC-ESI-FTICR analysis remains problematic since redistribution of excessive space charge trapped in an external rf-only trap is extremely complex and poorly predictable. Data-dependent control of the ion population in the external trap thus appears an attractive approach to expand the dynamic range of an FTICR instrument (e.g., by extending ion accumulation time for lower abundance peptides) and for eliminating m/z discrimination in the external rf-only trap (e.g., by shortening the ion accumulation times applied during LC elution of higher abundance peptides). Another undesired consequence of accumulating an excessive space charge in an external rf-only ion trap (e.g., a quadrupole), followed by efficient ion transfer and trapping in the ICR cell, is the increased cyclotron frequency shifts. FTICR can provide extremely high mass precisions and mass measurement accuracy (MMA),26,27 being best for trapping nearly constant, relatively small, and well-controlled ion populations in an ICR cell. However, protein concentrations of interest in proteomics vary by more than 10 000-fold28,29 and produce even larger variations in relative ion (19) Sannes-Lowery, K. A.; Hofstadler, S. A. J. Am. Soc. Mass Spectrom. 2000, 11, 1-7. (20) Hakansson, K.; Axelsson, J.; Palmblad, M.; Hakansson, P. J. Am. Soc. Mass Spectrom. 2000, 11, 210-215. (21) Belov, M. E.; Gorshkov, M. V.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2001, 12, 1312-1319. (22) Belov, M. E.; Gorshkov, M. V.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2000, 72, 2271-2279. (23) Belov, M. E.; Nikolaev, E. N.; Alving, K.; Smith, R. D. Rapid Commun. Mass Spectrom. 2001, 15, 1172-1180. (24) Belov, M. E.; Gorshkov, M. V.; Alving, K.; Smith, R. D. Rapid Commun. Mass Spectrom. 2001, 15, 1988-1996. (25) Gorshkov, M. V.; Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1993, 128, 47-60. (26) Gorshkov, M. V.; Masselon, C. D.; Anderson, G. A.; Udseth, H. R.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2001, 12, 11691173. (27) Qian, K.; Rodgers, R. P.; Hendrickson, C. L.; Emmett, M. R.; Marshall, A. G. 48th ASMS Conference on Mass Spectrometry and Allied Topics; Long Beach, CA, 2000.
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abundances at the peptide level. Therefore, the use of capillary LC separations of complex proteolytic digests in conjunction with FTICR poses a major challenge for accurate mass measurements. Given a fixed accumulation time in the external trap, the ratio of the total number of ions trapped in the ICR cell at different times during the LC separation can often span 2-3 orders of magnitude.11-13 Assuming that the space charge-related shift of the detected cyclotron frequency is directly proportional to the ion density and inversely proportional to the magnetic field strength,30 the uncorrected mass errors in identification of the same higher abundance peptide over its elution profile can be greater than 10 ppm (e.g., up to ∼ 25 ppm for a 3.5-T FTICR31), even if the same peptide could be detected with an error of 0.1 ppm at a particular low ion population in the ICR cell. As ion populations becomes larger, much more significant (and more difficult to correct) nonlinear frequency shifts occur.32 Therefore, a set of internal reference masses (i.e., calibrants) is generally required to correct for the space charge related frequency shifts. Francl et al. were able to demonstrate sub-ppm MMA for lowm/z ions by correlating the shift between an internal calibrant and the measured mass.30 Easterling et al. measured the protonated ion of insulin B-chain with an accuracy of 0.07 ppm using a polymer sample as an internal calibrant in MALDI-FTICR experiments.33 Using a similar approach, O’Connor and Costello introduced ions into an FTICR mass spectrometer from two separate MALDI samples, achieving MMA of
