Baseline Mass Resolution of Peptide Isobars: A Record for Molecular

Dec 30, 2000 - Baseline resolution of two peptides, RVMRGMR and RSHRGHR, of neutral monoisotopic mass, ∼904 Da, has been achieved by ...
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Anal. Chem. 2001, 73, 647-650

Baseline Mass Resolution of Peptide Isobars: A Record for Molecular Mass Resolution Fei He, Christopher L. Hendrickson, and Alan G. Marshall*,†

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

Baseline resolution of two peptides, RVMRGMR and RSHRGHR, of neutral monoisotopic mass, ∼904 Da, has been achieved by microelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at a mass resolving power of ∼3 300 000. The elemental compositions of these molecules differ by N4O vs S2H8 (0.000 45 Da), which is less than one electron’s mass (0.000 55 Da)! This result establishes a new record for the smallest resolved mass difference between any two molecules. This achievement is made possible by a combination of high magnetic field (9.4 T), large-diameter (4-in.) Penning trap, and low ion density. The implications for proteomics based on accurate mass measurements are discussed briefly. High mass resolving power, m/∆m50%, and high mass resolution (m2 - m1 g ∆m50%, in which m1 and m2 are the closest masses that can just be resolved, and ∆m50% is the mass spectral peak full width at half-maximum peak height) are analytically important for two reasons. First, as mass resolving power increases, so does the maximum number of components that may be distinguished in a complex mixture. Ultimately, it can become possible to resolve all chemically distinct components (except for isomers) in a mixture without prior separation. Second, at a given mass value, mass measurement accuracy increases with decreasing peak widthsat sufficiently high mass resolution (e.g., e0.001 Da), elemental composition (CcHhOoNn‚‚‚) may be determined uniquely from mass alone.1 Among all of the present methods for determining gas-phase ion mass, ion cyclotron resonance (ICR) in a Penning trap offers the highest mass resolving power and highest mass accuracy for ions of mass-to-charge ratio, m/z < 5000. Relative to ion beam methods (e.g., electric and/or magnetic sectors, linear quadrupoles, time of flight), ICR has the advantage that the cyclotron frequency (from which mass is determined) is nearly independent of ion kinetic energy and ion position during measurement. Relative to the quadrupole (Paul) ion trap, the ICR frequency is temporally more stable (because the magnetic field of a superconducting magnet is more stable than the magnitude of the rf voltage in a Paul trap) and is less sensitive to ion-neutral collisions and Coulomb interactions with other trapped ions. Finally, although atomic ion masses are typically determined with trapped † Member of the Department of Chemistry, Florida State University, Tallahassee, FL 32310. (1) Beynon, J. H.; Williams, A. E. Mass and Abundance Tables for Use in Mass Spectrometry; Elsevier: New York, 1963.

10.1021/ac000973h CCC: $20.00 Published on Web 12/30/2000

© 2001 American Chemical Society

ions of a single m/z value, molecular ion mass measurement requires calibration with ions of at least two m/z values and thus relies on Fourier transform ICR (FT-ICR) MS, as explained in detail elsewhere.2 High mass resolution (i.e., distinguishing ions of two closely spaced mass values) is much harder to achieve experimentally than the equivalent mass resolving power for ions of a single mass or two well-separated masses. For example, although mass resolving power up to m/∆m50% ≈ 200 000 000 was reported as far back as 1984 for singly charged Ar+ ions at ∼40 Da (namely, ∆m50% ≈ 0.000 000 2 Da),3 the best FT-ICR mass resolution for ions of two closely spaced m/z values in a single spectrum to date is (m2 - m1) ≈ 0.0009 Da for C6H479Br37Cl+ vs C6H481Br35Cl+ at ∼192 Da.4 The reason is that Coulomb repulsion between coherently orbiting packets of ions of different m/z can distort, shift, and ultimately coalesce their measured cyclotron resonances.5-8 The tendency of closely spaced ion cyclotron resonances to coalesce varies directly with ion mass and inversely with the difference in mass between the two ions.5 Thus, ICR mass resolution degrades rapidly with increasing ion mass. For example, although mass resolving power of g2 000 000 at up to ∼9 kDa has been reported previously,9,10 the isotopic “fine structure” (i.e., ions of the same nominal mass but different elemental composition) that should have been evident at that resolving power was not observed. Some years later, mass resolving power, m/∆m50% ≈ 5 000 000 (and ∆m50% < 0.003 Da), was achieved without peak coalescence at 16 kDa (p16 tumor suppressor protein), the highest mass at which isotopic “fine structure” has been resolved.11 However, the highest mass at which even “nominal” mass resolution (i.e., (m2 - m1) e 1 Da) has been achieved is 112 kDa (m/∆m50% ≈ 150 000, for chondroitinase enzyme).12 (2) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (3) Wanczek, K.-P. Pittsburgh Conf. Anal. Chem. Appl. Spectrosc., Atlantic City, NJ, 1987; Paper 526. (4) Hsu, C. S.; Liang, Z.; Campana, J. E. Anal. Chem. 1994, 66, 850-855. (5) Mitchell, D. W.; Smith, R. D. Phys. Rev. E 1995, 52, 4366-4386. (6) Mitchell, D. W.; Smith, R. D. J. Mass Spectrom. 1996, 31, 771-790. (7) Peurrung, A. J.; Kouzes, R. T. Int. J. Mass Spectrom. Ion Processes 1995, 145, 139-153. (8) Pasa-Tolic, L.; Huang, Y.; Guan, S.; Kim, H. S.; Marshall, A. G. J. Mass Spectrom. 1995, 30, 825-833. (9) Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Winger, B. E.; Smith, R. D. Rapid Commun. Mass Spectrom. 1993, 7, 700-703. (10) 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. (11) Shi, S. D.-H.; Hendrickson, C. L.; Marshall, A. G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11532-11537.

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As a special case, it is worth noting that if ions of opposite sign are trapped simultaneously, then it is possible to achieve FT-ICR mass resolution much better than the actual frequency resolution. For example, C60+ and C60-, differing by only the mass of two electrons, or ∼0.0011 Da, are easily resolved by digital quadrature heterodyne detection.13 Because of the difference in charge sign, the C60+ and C60- cyclotron frequencies are in fact very widely separated (by approximately twice the absolute value of either cyclotron frequency alone).14 However, this special situation does not address the much more typical problem of resolving ions (of the same charge sign) whose cyclotron frequencies are closely spaced. Finally, mass accuracy can exceed mass resolving power by a factor of up to ∼100. For any given ICR mass spectral peak shape, mass measurement imprecision (for a well-resolved peak) is proportional to the product of peak height-to-noise ratio and the square root of the number of data points per peak width (at, say, half-maximum peak height).15,16 In other words, mass may be measured to an imprecision much less than the peak width, provided that the signal-to-noise ratio is sufficiently high and there are enough data points per peak width. For example, the atomic mass of 20Ne has been measured by electron ionization FT-ICR MS as 19.992 440 691 (90) u (i.e., ∼5 ppb) at a mass resolving power of ∼20 000 000 (i.e., to within one-tenth of the peak width).17 With single-ion ICR techniques, atomic ion masses have been measured at sub-ppb accuracy.18 However, the present issue is mass resolution, not mass resolving power or mass accuracy, and we are interested in molecular, not atomic ions. In this paper, we report the electrospray FT-ICR mass spectra of two peptides, whose primary sequences were chosen to differ by (m2 - m1) < 0.0005 Da at ∼904 Da monoisotopic mass. We demonstrate baseline resolution of their ion cyclotron resonances, establishing a new record (by a factor of 2, at almost 5 times higher mass but doubly instead of singly charged ions) for the smallest molecular mass difference ever resolved. EXPERIMENTAL SECTION The two peptides, RVMRGMR and RSHRGHR, were synthesized by the BASS facility at Florida State University and used without further purification. The peptides were dissolved in 48: 48:4 water/MeOH/acetic acid to concentrations of 10 µM each and injected by constant infusion at a flow rate of 300 nL/min. Ions were generated by microelectrospray19,20 and detected with (12) Kelleher, N. L.; Senko, M. W.; Siegel, M. M.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1997, 8, 380-383. (13) Drader, J. J.; Shi, S. D.-H.; Blakney, G. T.; Hendrickson, C. L.; Laude, D. A.; Marshall, A. G. Anal. Chem. 1999, 71, 4758-4763. (14) Drader, J. J.; Schweikhard, L.; Shi, S. D.-H.; Hendrickson, C. L.; Marshall, A. G. 50th Pittsburgh Conf. Analytical Chem. and Appl. Spectroscopy, Orlando, FL, 1999; Abstr 2065P. (15) Chen, L.; Cottrell, C. E.; Marshall, A. G. Chemom. Intell. Lab. Syst. 1986, 1, 51-58. (16) Marshall, A. G.; Verdun, F. R. Fourier Transforms in NMR, Optical, and Mass Spectrometry: A User’s Handbook; Elsevier: Amsterdam, 1990. (17) Gorshkov, M. V.; Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1993, 128, 47-60. (18) DiFillip, F.; Natarajan, V.; Bradley, M.; Palmer, F.; Pritchard, D. E. Phys. Scr. 1995, T59, 144-154. (19) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (20) Emmett, M. R.; White, F. M.; Hendrickson, C. L.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998, 9, 333-340.

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a 9.4-T home-built FT-ICR mass spectrometer21 equipped for external ion accumulation22 and controlled by a modular ICR data acquisition system (“MIDAS”).23 Ions of undesired m/z values were removed by SWIFT dipolar ejection before detection.24,25 Following SWIFT isolation, the trapping potential on each end cap electrode was dropped stepwise from 4 to 0.14 V in a total of 10 steps over a period of ∼60 s.11 (It is easiest to tune the experiment by starting with a large number of (coalesced) ions and then slowly decrease the ion population until the species are resolved. In that way, there is positive feedback (i.e., detectable signal) on every scan. The alternative would be to target initially a small ion population and tune toward more signal based on negative feedback (i.e., absence of signal)). After the ions had cooled, they were detected in digital quadrature heterodyne mode13 at a reference frequency of 317.4 kHz and a sampling frequency of 20 kHz for 26 s to yield 512 Kword complex timedomain data. After padding with another 512K zeroes, fast Fourier transformation (with Hanning apodization16) was followed by magnitude calculation, and frequency was converted to mass-tocharge ratio by the standard quadrupolar trapping potential approximation.26 The spectral bandwidth of 10 kHz results in an FT-ICR frequency-domain point spacing of ∼0.019 Hz, corresponding to an FT-ICR mass spectral point spacing of ∼0.000 054 Da for a doubly charged ion at m/z 453. Although each displayed spectrum resulted from a single excitation/detection event sequence (i.e., no signal-averaging), the experiment was repeated several times with the same result to establish reproducibility. Finally, the measured pressure in the pumping stage prior to the cell is ∼9 × 10-10 Torr, so that the pressure at the cell cannot differ much from the pressure reading (∼5 × 10-10 Torr) from an ion gauge located ∼1 m from the cell. Thus, the FT-ICR mass spectral peak width is not pressure limited. RESULTS AND DISCUSSION The two peptides, RVMRGMR and RSHRGHR differ by three amino acids, VMM vs SHH. Their elemental compositions (C35H68N16O8S2 vs C35H60N20O9) differ by S2H8 vs N4O, for a mass difference of ∼0.000 453 Da (which is less than the electron mass (0.000 549 Da). Figure 1 shows a microelectrospray FT-ICR directmode (broadband) mass-to-charge ratio spectrum of the two equimolar peptides. Doubly charged (M+2H)2+ ions of both peptides were isolated by SWIFT ejection and yielded wellresolved isotopic distributions (Figure 1, inset). However, the discrete point spacing in that spectrum (0.0017 Da) is larger than the mass separation (0.000 453 Da) between the two peptides at a given nominal mass, so that the two peptides cannot be resolved. Therefore, digital quadrature heterodyne (narrowband) detection13 was performed on the peptide isotope distributions shown (21) Senko, M. W.; Hendrickson, C. L.; Pasa-Tolic, L.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828. (22) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (23) Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1839-1844. (24) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (25) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 5-37. (26) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 27442748.

Figure 1. Electrospray 9.4-T FT-ICR mass-to-charge ratio spectrum of two equimolar peptides, RVMRGMR and RSHRGHR. The doubly charged ions were isolated by stored-waveform inverse Fourier transform (SWIFT) dipolar ejection. Although isotopic peaks are well resolved (see mass scale-expanded inset), the two peptides are not resolved from each other.

Figure 2. Ultrahigh-resolution ESI FT-ICR mass spectrum of monoisotopic (M+2H)+ ions (labeled by an asterisk in Figure 1) detected in digital quadrature heterodyne (narrowband) mode. The two peptides differing in mass by 0.000 45 Da are baseline-resolved. The achieved resolving power is m/∆m50% ) 3 300 000, in which ∆m50% is the magnitude-mode peak full width at half-maximum peak height.

in Figure 1. to yield baseline resolution of the two peptides, as shown in Figure 2. The experimentally measured mass difference between the two peaks is measured as 0.000 452 Da, in agreement with the true value to within 1 µDa, and an improvement by a factor of 2 over the closest previously resolved molecular mass doublet.4 At the experimental mass resolving power (m/∆m50% ≈ 3 300 000) shown in Figure 2, the experimental peak width (∆m50%) is ∼0.000 27 Da) so that that two peptides are separated by ∼0.6 line width (i.e., sufficient for baseline resolution). To eliminate any possibility that the observed two peaks arise from artifacts such as peak splitting induced by imperfect electrostatic field in the ICR trap,27,28 or even noise spikes, ESI FT-ICR mass spectra were acquired from solutions containing only one of either peptide under the same experimental conditions. For either peptide, a single peak was observed at the same m/z (27) Rempel, D. L.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1992, 3, 590-594. (28) Jackson, G. S.; White, F. M.; Guan, S.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1999, 10, 759-769.

value, at comparable resolving power, as when both peptides were present. The signal magnitudes from the (equimolar) peptides are comparable (to within the baseline electronic noise level), and any small remaining difference likely results from different electrospray efficiency as well as statistical fluctuation in the relatively small number of ions. The present ultrahigh mass resolution may be attributed to several combined factors: high magnetic field, B0 ) 9.4 T, since ICR mass resolving power varies directly with B0;29 large ion trap diameter, (d ) 10 cm), since ICR frequency coalescence tendency varies inversely with ion excitation radius; 5 and digital quadrature heterodyne detection, which improves signal-to-noise ratio by a factor of 21/2,13 since a given S/N ratio requires 21/2 fewer ions and peak coalescence scales with ion number. In addition, extended cooling of ions prior to detection produces low ion density, which reduces peak shifts, distortion, and/or coalescence. To cool the ions, we reduced the cell trapping potential stepwise,11,30 thereby allowing translationally hot ions to evaporate and also allowing ions to spread out axially for reduced Coulomb interactions. CONCLUSIONS AND IMPLICATIONS Here, we have resolved two peptides differing in mass by less than 0.0005 Da. However, for analysis of actual tryptic (or other enzymatic) protein digests, it will not be feasible to perform heterodyne detection separately for each potential mass doublet, as in Figure 2. The more relevant practical questions for proteomics are as follows: (a) what mass resolution can be achieved routinely by “direct” (broadband) detection over a wide mass range; (b) what mass resolution and accuracy are needed to be able to identify a peptide or protein uniquely? With respect to experimental mass resolution, ESI FT-ICR MS at 9.4 T in direct (broadband) detection mode routinely resolves peptides separated by 0.005 Da (with correspondingly high mass accuracy) at ∼1000 Da in a mixture of dozens of tryptic peptides. Unique identification of the amino acid composition of a 1000 Da peptide is not feasible if all possible amino acid compositions are considered, because multiple peptides can have the same elemental formula.31 In proteomics, one usually faces the much less restrictive issue of choosing the correct peptide sequence from a much smaller number of known proteins (e.g., ∼85 000 proteins from the SWISS-PROT database).32 As we shall further delineate in a full paper, the number of candidate proteins identified by accurate measurement of, for example, tryptic peptide masses decreases dramatically (to as few as one protein based on three tryptic fragments) if peptide masses can be resolved and measured to within less than ∼0.005 Da33 (i.e., a mass measurement accuracy achievable only with FT-ICR MS). It is important to distinguish (29) Marshall, A. G.; Guan, S. Rapid Commun. Mass Spectrom. 1996, 10, 18191823. (30) Li, G. Z.; Poggiani, R.; Testera, G.; Werth, G. Z. Phys. D 1991, 22, 375382. (31) Zubarev, R. A.; Hakansson, P.; Sundqvist, B. Anal. Chem. 1996, 68, 40604063. (32) Bairoch, A.; Apweiler, R. Nucleic Acids Res. 2000, 28, 45-48. (33) van der Rest, G.; He, F.; Marshall, A. G.; Hubbard, S.; Gaskell, S. 48th Am. Soc. Mass Spectrom. Annu. Conf. Mass Spectrom. Allied Top., Long Beach, CA, 2000; Abstr. WP225.

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the present performance from prior MALDI-TOF experimentsperformed at a much lower mass resolving power of 5000-10 000,34 for which the peak centroid must be determined to within a few percent of the peak width (i.e., very high abundance of ions of that mass-to-charge ratio35) and there must be no overlap with other species within ∼100-200 mDa in mass (e.g., Lys vs Gln, a common doublet separated by 36 mDa). The only way to be sure of a mass assignment is to be able to resolve the species of interest (34) Tackach, E. J.; Hines, W. M.; Patterson, D. H.; Juhasz, P.; Falick, A. M.; Vestal, M. L.; Martin, S. A. J. Protein Chem. 1997, 16, 363-369. (35) Lee, H.-N.; Marshall, A. G. Anal. Chem. 2000, 72, 2256-2260.

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from other species of nearly the same mass, as in the present approach. ACKNOWLEDGMENT This work was supported by grants from the NSF National High Field FT-ICR Mass Spectrometry Facility (CHE-99-09502), NIH (GM-31683), Florida State University, and the National High Magnetic Field Laboratory in Tallahassee, Florida. Received for review August 15, 2000. Accepted November 20, 2000. AC000973H