Simplification of Product Ion Spectra Derived from Multiply Charged

High-mass multiply charged ions fragment to yield a mixture of products of varying mass and charge. When the measurement of mass-to-charge ratio is us...
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Anal. Chem. 1998, 70, 3533-3544

Simplification of Product Ion Spectra Derived from Multiply Charged Parent Ions via Ion/Ion Chemistry James L. Stephenson, Jr., and Scott A. McLuckey*

Chemical and Analytical Sciences Division Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365

High-mass multiply charged ions fragment to yield a mixture of products of varying mass and charge. When the measurement of mass-to-charge ratio is used to determine product ion mass, product ion charge must first be established. To minimize charge-state ambiguity in product ion spectra derived from multiply charged parent ions, product ions have been subjected to protontransfer reactions with oppositely charged ions to reduce product ion charge states largely to +1. This procedure greatly simplifies the interpretation of product ion spectra derived from multiply charged ions. Illustrative data are presented for the +4 and +3 parent ions derived from electrospray of melittin and the +12 to +4 parent ions of bovine ubiquitin, whereby product ions were formed in a conventional quadrupole ion trap tandem mass spectrometry experiment. Data are also shown for product ion mixtures derived from interface-induced dissociation of multiply charged ions derived from bovine ubiquitin, tuna cytochrome c, bovine cytochrome c, and equine cytochrome c. The use of ion/ion chemistry to simplify product ion spectra derived from multiply charged parent ions significantly extends the size range of macromolecules for which the quadrupole ion trap can derive structural information. With the advent of electrospray ionization,1-4 multiply charged ions derived from polymeric species have become commonplace in analytical mass spectrometry. The phenomenon of multiple charging has been exploited for the mass measurement of highmolecular-weight species using mass analyzers of relatively modest mass-to-charge range. Furthermore, it has been noted that multiple charging facilitates the dissociation of high-mass ions5-10 thereby allowing for the determination of structural * Corresponding author: (phone) (423) 574-2848; (fax) (423) 576-8559; (email) [email protected]. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (3) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882-899. (4) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451. (5) Busman, M.; Rockwood, A. L.; Smith, R. D. J. Phys. Chem. 1992, 96, 23972400. (6) Rockwood, A. L.; Busman, M.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 582-585. (7) Rockwood, A. L.; Busman, M.; Smith, R. D. Int. J. Mass Spectrom. Ion Processes 1991, 111, 103-129. S0003-2700(98)00283-2 CCC: $15.00 Published on Web 07/30/1998

© 1998 American Chemical Society

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)11 have been generated with commonly employed instruments, such as the triple-quadrupole tandem mass spectrometer. A variety of factors can lead to the ready dissociation of highly charged ions, including reduced kinetic stabilities of the ions at high charge as a result of changes in dissociation mechanism and/or dissociation threshold, and, in the case of collisional activation, higher collision energies obtainable with highly charged ions. Furthermore, it is commonly observed that the identities and relative abundances of product ions from multiply charged biopolymers can be highly dependent upon the charge state of the parent ion.3,9-14 Therefore, complementary primary structure information is often obtained from the product ions derived from several of the charge states of a biopolymer. For these reasons, the multiple-charging phenomenon associated with electrospray of biopolymers can be regarded as highly desirable for obtaining primary structure information. 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. Unlike the electrospray mass spectrum, in which there is typically a distribution of coherent charge states, the product ions do not necessarily show multiple-charge states for the same product ion. Algorithms commonly used to convert electrospray mass spectra to “zero-charge” spectra15-21 rely on (8) Ishikawa, K.; Nishimura, T.; Koga, Y.; Niwa, Y. Rapid Commun. Mass Spectrom. 1994, 8, 933-938. (9) Ve´key, K. Mass Spectrom. Rev. 1995, 14, 195-225. (10) Price, W. D.; Schnier, P. D.; Williams, E. R. Anal. Chem. 1996, 68, 859866. (11) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991, 63, 24882499. (12) Loo, J. A.; Loo, R. R. O.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101-105. (13) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1993, 65, 425-438. (14) McLuckey, S. A.; Habibi-Goudarzi, S. J. Am. Chem. Soc. 1993, 115, 1208512095. (15) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702-1708. (16) Ferrige, A. G.; Seddon, M. J.; Jarvis, S. Rapid Commun. Mass Spectrom. 1991, 5, 374-379. (17) Ferrige, A. G.; Seddon, M. J.; Green, B. N.; Jarvis, S. A.; Skilling, J. Rapid Commun. Mass Spectrom. 1992, 6, 707-711. (18) Reinhold, B. B.; Reinhold: V. N. J. Am. Soc. Mass Spectrom. 1992, 3, 207215. (19) Labowsky, M.; Whitehouse, C.; Fenn, J. B. Rapid Commun. Mass Spectrom. 1993, 7, 71-84. (20) Hagen, J. J.; Monnig, C. A. Anal. Chem. 1994, 66, 1877-1883.

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the appearance of ions with at least two charge states for each component in the spectrum and therefore have limited utility for spectra of fragments resulting from dissociation of multiply charged ions (i.e., product ion spectra). A number of measures have been developed to address the determination of charge states in product ion spectra derived from multiply charged parent ions. A useful, but not entirely reliable strategy for inferring product ion charge states is to identify the complementary ions produced from a dissociation that leads to two charged products.13,22 A definitive approach, provided the resolving power of the mass analyzer is sufficiently high, is to measure the spacings of the isotope peaks in the product ion.23 The fractional mass-to-charge spacings of adjacent isotopes yield the charge-state information directly. There are several other examples of the measurement of peak spacings for product ion charge-state determination. For example, Senko et al. described the measurement of copper adduct ion spacings present in product ions for the facilitation of charge determination using a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer.24 When the spacings between peaks with known mass or charge relationships are significantly greater than the spacings between the isotope peaks, as in the study just mentioned, analyzers of much lower resolving powers than high magnetic field strength FTICR25 can be used to determine product ion charge states. For example, we have described the use of strong gaseous neutral bases to promote either proton transfer or adduct formation in a quadrupole ion trap as a means for generating two ions with a known charge or mass relationship.26 In this way the mass-to-charge ratios of the two ions can be used to determine their charges. Hunter et al. have demonstrated the use of ion/molecule proton-transfer reactions for charge-state determination as well using a triple-quadrupole mass spectrometer.27 We have also reported on the use of singly charged ions of polarity opposite to that of the product ion of interest to generate two ions with a known mass and charge relationship28 (i.e., ∆m ) 1 Da and ∆z ) 1). In both of the ion trap reports,26,28 the methodology for determining product ion charge states involved MS3 experiments whereby the mass selected parent ion was dissociated and a product ion of interest was then isolated and subjected to either an ion/molecule or ion/ion reaction. While it has been shown that ion/ion reactions are more universal than ion/molecule reactions for reducing charge states, the use of an MS3 experiment for each product ion is a relatively tedious and inefficient process. All of the approaches mentioned above for the determination of product ion charge states rely on the measurement of the massto-charge spacings between two or more ions related in some fashion to the species of interest. The spacings may differ by a (21) Zhang, Z.; Marshall, A. G. J. Am. Soc. Mass Spectrom, in press. (22) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Science 1990, 248, 201-204. (23) Henry, K. D.; McLafferty, F. W. Org. Mass Spectrom. 1990, 25, 490-492. (24) Senko, M. W.; Beu, S. C.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1993, 4, 828-830. (25) Marshall, A. G.; Guan, S. Rapid Commun. Mass Spectrom. 1996, 10, 18191823. (26) McLuckey, S. A.; Glish, G. L.; Van Berkel, G. J. Anal. Chem. 1991, 63, 1971-1978. (27) Hunter, A. P.; Severs, J. C.; Harris, F. M.; Games, D. E. Rapid Commun. Mass Spectrom. 1994, 8, 417-422. (28) Herron, W. J.; Goeringer, D. E.; McLuckey, S. A. Anal. Chem. 1996, 68, 257-262.

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known mass or by a known difference in mass and charge. Recent studies in our laboratory based on the use of ion/ion protontransfer chemistry for the analysis of ions derived from protein mixtures29,30 have indicated that a simple approach might be implemented to facilitate the interpretation of product ion spectra derived from multiply charged ions that does not require the measurement of two related peaks. The approach involves subjecting the entire product ion population to ion/ion reactions, which leads to a product ion spectrum in which singly charged ions dominate so that charge-state ambiguities are minimized. This approach does not require MS3 procedures and therefore allows the masses of the all of the product ions to be determined following a single MS/MS experiment. The likelihood that such a strategy would be effective was made apparent from our observation that the kinetics of ion/ion proton-transfer reactions are amenable to the use of a single ion/ion reaction period to reduce ions predominantly to singly charged species independent of the initial charge state.29-31 We report here our observations with product ion mixtures derived from the dissociation of ions formed by electrospray of melittin, bovine ubiquitin, and several forms of cytochrome c. EXPERIMENTAL SECTION Melittin, bovine ubiquitin, and tuna, bovine, and equine cytochrome c were obtained from Sigma Chemical Co., St. Louis, MO. Perfluoro-1,3-dimethylcyclohexane (PDCH) was purchased from Aldrich, Milwaukee, WI. Solutions for electrospray were prepared by dissolving the sample in 1 mL to give a concentration of 1-10 µM in 50:50 methanol/water (v/v). Acetic acid was added to 1% for all protein solutions. All solutions were infused at a rate of 1.0 µL/min through a 50-µm-i.d. fused-silica needle. Electrical contact was made at a metal fitting upstream of the fused silica with an applied positive potential of 3.0-4.0 kV. The experiments were performed using a Finnigan ion trap mass spectrometer modified for electrospray to allow for ion injection through an end cap electrode, and glow discharge32 for ion injection through a hole in the ring electrode.33 All experiments were controlled by ICMS software.34 The typical sequence of events involving the dissociation of a selected ion charge state was as follows: positive ion accumulation, isolation of a charge state of interest, collisional activation of the isolated parent ion, anion accumulation, mutual storage of negative ions and positive ions, ejection of negative ions, and mass analysis of positive ions. For experiments in which product ions were formed in the electrospray interface, the ion isolation and ion activation steps just described were eliminated. Positive ions formed by electrospray were injected into the ion trap over a period of 0.3-0.4 s. The radio frequency (rf) sine wave amplitude applied to the ring electrode during ion injection was 865-1040 V (0-p) (ring electrode radius 1.0 cm, drive frequency 1.1 MHz). In all cases, (29) McLuckey, S. A.; Stephenson, J. L., Jr.; Asano, K. G. Anal. Chem. 1998, 70, 1198-1202. (30) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1998, 9, 585-596.. (31) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Chem. Soc. 1996, 118, 73907397. (32) McLuckey, S. A.; Glish, G. L.; Asano, K. G.; Grant, B. C. Anal. Chem. 1988, 60, 2220-2228. (33) Stephenson, J. L., Jr.; McLuckey, S. A. Int. J. Mass Spectrom. Ion Processes 1997, 162, 89-106. (34) ICMS software provided by N. Yates and the University of Florida.

helium was admitted into the vacuum system to a total pressure of roughly 1 mTorr with a background pressure in the instrument of 2 × 10-5 Torr without the addition of helium. Negative ions were formed by sampling the headspace vapors of PDCH into the glow discharge operated at 800 mTorr. The discharge was gated via software control so that it was on only during the anion accumulation period to avoid detector noise observed when the discharge is operated continuously.35,36 Negative ion accumulation times were typically 20-45 ms with an rf amplitude applied to the ring electrode during negative ion accumulation of 577 V (0-p). Negative ion/positive ion mutual storage times were 100120 ms, and in all cases, the rf amplitude applied to the ring electrode during this period was 1730 V. Negative ions were ejected at the end of the mutual storage period to avoid deleterious effects on mass analysis of the positive ions resulting from the electric field created by stored negative ions.37 Unless otherwise noted, negative ion ejection was effected by a 10-ms ramp of the amplitude of the drive rf to eject ions of mass/charge ratio up to about 620. Parent ion isolation was effected with two resonance ejection ramps.38 The first 30-ms ramp consisted of a sweep of the rf amplitude applied to the ring electrode from 1150 to 7500 V (0-p) while a 6-V peak-to-peak sine wave was applied to the end cap electrodes. The frequency of this sine wave was selected to eject ions of mass-to-charge less than that of the parent ion. The second ramp was identical except that the frequency of the sine wave applied to the end caps was changed to eject ions of higher mass-to-charge ratio than that of the parent ion. This procedure yields a window of mass-to-charge ratio in which the ions of interest remain in the ion trap. The width of this window is defined by the frequencies applied to the end caps and their amplitudes. For these studies, the window was kept sufficiently wide to avoid any noticeable parent ion ejection as a result of offresonance power absorption. The isolated window was therefore wider than the mass-to-charge range of the ions that were subjected to resonance excitation (see below). Ion activation was performed using single-frequency resonance excitation.39,40 In all cases, the parent ions were activated at a qz value of 0.25 (z-dimension fundamental frequency of motion of about 96 kHz). It was noted that the best product ion peak shapes were observed using low resonance excitation amplitudes (100200 mV) and long activation times (100-500 ms). This observation has been attributed to there being less sequential fragmentation and, perhaps, fewer dissociation channels at low amplitudes and long times than at higher amplitudes (400-800 mV mV) and short excitation times (10-30 ms). Furthermore, there is less likelihood for parent ion ejection at the lower amplitudes. These observations are expected based on the slow heating nature of ion trap collisional activation.41,42 The lower amplitude activation (35) McLuckey, S. A.; Goeringer, D. E.; Asano, K. G.; Vaidyanathan, G.; Stephenson, J. L., Jr. Rapid Commun. Mass Spectrom. 1996, 10, 287-298. (36) Duckworth, D. C.; Smith, D. H.; McLuckey, S. A. J. Anal. At. Spectrosc. 1997, 12, 43-48. (37) Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 1997, 69, 3760-3766. (38) McLuckey, S. A.; Goeringer, D. E.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1991, 2, 11-21. (39) 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. (40) Johnson, J. V.; Yost, R. A.; Kelley, P. E.; Bradford, D. C. Anal. Chem. 1990, 62, 2162-2172. (41) Goeringer, D. E.; McLuckey, S. A. J. Chem. Phys. 1996, 104, 2214-2221.

elevates the parent ions to lower temperatures than does higher amplitude activation, and the use of longer times allows for the accumulation of ions formed from lower rate processes. The use of low resonance excitation amplitudes, however, results in a relatively narrow effective frequency bandwidth for ion excitation. It was noted that irreproducibility from one scan to the next in the number of positive ions accumulated during the ion injection period resulted in relatively inconsistent ion activation. This situation was ameliorated somewhat by use of higher amplitudes (200-600 mV) at a resonance excitation frequency a few hundred hertz less than the optimum. We expect this procedure to be somewhat analogous to sustained off-resonance irradiation commonly used for collisional activation of ions in FTICR mass spectrometry.43,44 We did not use the much higher amplitudes (10-20 V) reported by Qin and Chait, in the so-called red-shifted off-resonance large-amplitude excitation approach,45 because it tends to eject a relatively wide range of product ions that fall just above the mass-to-charge ratio of the parent ion, a region where product ions are commonly formed from multiply charged parents. Mass analysis was effected via resonance ejection.46 For spectra acquired without use of ion/ion reactions, a resonance ejection frequency and amplitude of 89 202 kHz and 4.2 V (p-p), respectively, was used. This set of conditions provided an upper mass/charge limit of 2600. Data acquired after the ion/ion reactions were obtained using a resonance ejection amplitude of 3.4 V (p-p) and a frequency adjusted to provide an upper mass/ charge limit that encompassed the mass/charge of the singly charged parent ion (2846 for melittin, 8564 for ubiquitin, 12 029 for tuna cytochrome c, 12 231 for bovine cytochrome c, and 12 360 for equine cytochrome c). In all cases, the rate of change of the amplitude of the rf voltage applied to the ring electrode was the standard rate supplied by the ITMS electronics (128 V/ms). The mass/charge scales for the various spectra were calibrated using the electrospray mass spectra of the standards (melittin, bovine ubiquitin, and the cytochromes). These standards were checked by acquiring electrospray mass spectra with a PE-Sciex API 165 quadrupole mass analyzer calibrated with a solution of poly(propylene glycol). With the known masses of the standards, the post-ion/ion MS/MS spectra could be calibrated using ion/ion reaction data collected in the absence of collisional activation. All spectra were acquired using the same voltages applied to the electron multiplier detector and conversion dynode. The postion/ion reaction spectra derived from mass-selected parent ions were typically the average of 400 scans. The post-ion/ion reaction spectra derived from interface-induced dissociation were the average of 200 scans. The pre-ion/ion reaction spectra were typically the average of 100 scans. While mass determination of the product ions could be made with a factor of 10 or fewer scans, the data were extensively averaged in this work so that reliable observations might be drawn regarding the relative abundances of the product ions observed in the pre- and post-ion/ion reaction data. (42) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 32, 461-474. (43) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (44) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012809. (45) Qin, J.; Chait, B. T. Anal. Chem. 1996, 68, 2108-2112. (46) Kaiser, R. E., Jr.; Cooks, R. G.; Moss, J.; Hemberger, P. H. Rapid Commun. Mass Spectrom. 1989, 3, 50-53.

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RESULTS AND DISCUSSION In considering the data presented below, it is useful to review briefly what has already been learned about the reactions of the anions derived from PDCH with multiply protonated biomolecules. Glow discharge ionization of PDCH and injection of the negative ions through the ring electrode using an rf drive amplitude of 577 V (0-p) yields predominantly the (M - F)- and (M - CF3)ions.33 The only reaction that has yet been observed for the reactions of these ions with multiply protonated polypeptides has been proton transfer.47,48 We have seen no evidence so far for attachment of these anions to the cations, fluoride transfer from these anions to polypeptide cations, or fragmentation of the positive polypeptide ions as a result of ion/ion reaction. Some of the reaction phenomenologies just mentioned have been observed with other combinations of cations and anions49 but not with anions derived from PDCH and cations derived from biopolymers. Furthermore, we have found no evidence for anything other than proton transfer in the reactions of the polypeptide product ions in this study. That is, there is no evidence for the appearance of any new products as a result of ion/ion chemistry other than those resulting from charge-state reduction. We have commented previously on the kinetics of the reactions of multiply charged ions with ions of opposite polarity in the ion trap.29-31 For the purpose of this study, it is important to recognize that the most important parameter in determining the relative rates of ion/ion reactions for a mixture of positive ions composed of various numbers of charges is the cation charge. We have found it to be useful to model the time evolution of positive ions undergoing reactions with an excess of negative ions as a set of irreversible consecutive reactions48 in which the rate constant for a given change in unit charge is

kc ) νπ[z1z2e2/(µν2)]2

(1)

where ν is the relative velocity of the oppositely charged ions, µ is the reduced mass of the collision partners, and z1 and z2 are the charges of the cations and anions, respectively. Due to uncertainty in the anion number density, we have not demonstrated that eq 1 provides a quantitative prediction of the ion/ion reaction rate constant. However, we have observed that, with a constant anion number density, reaction rates follow the predicted z12 dependence.31 Ion mass is the other parameter affecting relative rates, via the reduced mass term in the denominator of eq 1, but it has only a minor influence relative to ion charge. An important consequence of the primary dependence of reaction rates on ion charge is that singly charged ions react slowly relative to ions of higher charge states such that a significant fraction, typically one- to two-thirds, of the singly charged ions present prior to the ion/ion reaction period do not undergo neutralization while the more highly charged ions in the mixture are reduced to a single charge. Therefore, the relative abundances of the dissociation products, irrespective of charge, after ion/ion reaction are not expected to be changed by more than a factor of 2-3 from (47) Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 1996, 68, 4026-4032. (48) Stephenson, J. L., Jr.; Van Berkel, G. J.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1997, 8, 637-644. (49) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Chem. Soc. 1997, 119, 16881696.

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their pre-ion/ion reaction relative abundances due to the ion/ion reaction kinetics. The largest changes in relative product ion abundances in the pre- versus post-ion/ion reaction data are expected to be observed when the products initially present at the two charge-state extremes are compared (i.e., singly charged product ions versus product ions of charge equal to that of the parent ion). Any changes greater than a factor of 2-3 must arise from some other source of discrimination (see below). The single-reaction type (i.e., proton transfer) and the chargesquared dependence of the rates for ion/ion reactions involving polypeptide cations and PDCH anions have important implications for this application. First, ion/ion chemistry adds no new products to the product ion spectrum, and second, ion/ion chemistry does not eliminate any products over these time frames. The major perturbation introduced by the use of ion/ion chemistry to simplify the product ion spectrum is that there may be relatively modest changes in the relative abundances of the products due to somewhat different degrees of total neutralization. The greatest degree of discrimination is expected to be against products initially present as singly charged ions. Melittin. Product ion spectra of multiply charged melittin ions derived from several methods and using a variety of tandem mass spectrometers have been reported.5,50-54 This system provides some relatively small, well-studied multiply charged ions wherein the identities of the product ions are already established. Triply and quadruply protonated melittin constitute the most abundant ions derived from electrospray with the present instrument. The ion trap collisional activation and ion/ion reaction data are described here for both. Figure 1 compares the normal ion trap MS/MS spectrum of the (M + 4H)4+ ion of melittin (a) with the spectrum acquired after the product ions reflected in Figure 1a were subjected to ion/ion reactions for 100 ms. Figure 1a is typical of MS/MS spectra derived from multiply charged parent ions in that there is a mixture of product ion charge states extending, in this case, from +1 to +4. The major quadruply charged product ions correspond to losses of water and/or ammonia. All of the other major product ions can be accounted for as either b-type or y-type ions, according to the standard nomenclature,55 with one, two, or three charges. The most intense product ions arise from the commonly observed cleavage at proline and correspond to y133+ and y132+. Figure 1b shows how the appearance of the spectrum changes when the product ion mixture is subjected to ion/ion proton-transfer reactions for a period sufficient to drive most of the ions to the +1 charge state. In this case, potential chargestate overlap is eliminated in the mass/charge region greater than half the mass of the singly charged parent ion. All ions appearing in this region must be singly charged. Potential remains for charge-state overlap below half-mass. However, when the reaction (50) Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1989, 3, 160-164. (51) Van Berkel, G. J.; McLuckey, S. A.; Glish, G. L. Anal. Chem. 1990, 62, 1284-1295. (52) Fabris, D.; Kelly, M.; Wu, Z.; Fenselau, C. Rapid Commun. Mass Spectrom. 1994, 8, 791-795. (53) Meot-Ner, M.; Dongre´, A. R.; Somogyi, AÄ .; Wysocki, V. H. Rapid Commun. Mass Spectrom. 1995, 9, 829-836. (54) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (55) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601.

Figure 1. (a) Pre-ion/ion reaction MS/MS spectrum of the (M + 4H)4+ parent ion of melittin. (b) Post-ion/ion reaction MS/MS spectrum of the (M + 4H)4+ parent ion of melittin.

time is chosen to yield largely singly charged ions, there should be no doubly charged ions without a singly charged ion also present in the spectrum. This greatly facilitates the identification of doubly charged ions in the post-ion/ion reaction data. While the signal level of the y13 product (both as the +2 and +1 ions) dominates the post-ion/ion reaction spectrum, as it does in the pre-ion/ion reaction spectrum (as the +3 and +2 ions), signals can be readily identified for the b-series ions b7-b13 and for the y-series ions y5, y8-y22, and y24. Evidence for all of the products identified in the post-ion/ion reaction spectrum can be found in the pre-ion/ion reaction data, but in the absence of some means for clearly establishing charge state, assumptions about ion charge are needed to fully interpret the pre-ion/ion reaction spectrum. Furthermore, there are no products identified in the pre-ion/ion spectrum that are missing in the post-ion/ion reaction data, aside from those that may have been ejected by the negative ion ejection ramp. Figure 2 compares the product ion spectra derived from the (M + 3H)3+ parent ion both before (Figure 2a) and after (Figure 2b) ion/ion proton-transfer reactions. This parent ion offers little in the way of sequence information that cannot be obtained from the product ion spectrum of the (M + 4H)4+ ion, but the relative contributions of the various competitive dissociation channels differ markedly. The most obvious difference is the much lower relative contribution from the channel leading to the y13 fragment.

Figure 2. (a) Pre-ion/ion reaction MS/MS spectrum of the (M + 3H)3+ parent ion of melittin. (b) Post-ion/ion reaction MS/MS spectrum of the (M + 3H)3+ parent ion of melittin.

Several features of the post-ion/ion reaction data illustrate additional points regarding the use of ion/ion chemistry for simplifying the interpretation of product ion spectra. First, fewer anions were used to collect the data of Figure 2b than for the data of Figure 1b (20- versus 45-ms anion accumulation). Therefore, the relative contribution of doubly charged ions in the mass/charge region less than 1400 is significantly greater in Figure 2b than it is in Figure 1b. This comparison (viz., the relative contributions of doubly charged ions in Figures 1b and 2b), highlights a tradeoff that must be made between total product ion signal and the extent to which the post-ion/ion reaction product ion spectrum is dominated by singly charged ions. Use of relatively few anions or, equivalently, a relatively short ion/ion reaction time can minimize the number of product ions that are completely neutralized. However, it also increases the possibility for charge-state overlap in the mass/charge region less than half the mass/charge of the singly charged parent ion. It is not possible to generate a product ion spectrum in which over 90% of all of the ions in the product ion spectrum are singly charged without neutralizing a significant fraction of the ions (i.e., several tens of percent). On the other hand, if there is sufficient structural information present above half the m/z ratio of the singly charged ion, the reaction time (or number of anions) can be selected to maximize the signals of the ions that fall within this mass/charge range. The Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

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ion/ion reaction time or, equivalently, the number of anions can be regarded as variables in optimizing the MS/MS strategy used either to identify or to sequence a polypeptide. The data acquired for the (M + 3H)3+ parent ion of melittin also illustrate the frequently encountered situation in which a product ion falls within the mass/charge window selected for isolation of the parent ion. The window selected to isolate the parent ion fell around m/z 950. After ion activation, it is clear that there are several signals overlapping in this region in the pre-ion/ion reaction spectrum (Figure 2a). This signal is composed of undissociated parent ions, ions formed from losses of water and/or ammonia, undissociated ions unrelated to melittin that happened to fall within the ion isolation window, and any product ions that happen to have an m/z ratio that falls within this region. Provided the amplitude of the signal used to dissociate the parent ion is insufficient to eject all product ions that happen to fall into the selected m/z window, the product ion should be accumulated (and, perhaps, itself be activated). In this case, the y162+ ion is an example of a product ion that falls near in m/z to that of the parent ion. The MS/MS data of the (M + 3H)3+ parent ion alone would not allow for the identification of the y16 fragment and would therefore not allow for the identification of the threonine residue at that location. The post-ion/ion reaction data (Figure 2b) shows the peaks arising from water and/ or ammonia loss from the parent ion, the undissociated singly charged parent ion, an abundant y16 fragment, and a peak possibly arising from water and/or ammonia loss from the y16 fragment clearly separated from one another. Note the lower abundance of the y16 fragment relative to the y15 and y17 fragments and the larger water or ammonia loss peak associated with this fragment relative to the other y-series fragments. These observations may arise from the ejection/activation of some of the doubly charged y16 product ions by the activation signal intended to dissociate the parent ion. Further studies would be required to determine the extent to which ejection/activation of the product ions takes place in situations such as this. In any case, the comparison of Figure 2 clearly shows that it is straightforward to separate product ions from ions of different charge present in the isolated parent ion mass/charge window after collisional activation. Figure 2 also illustrates a common observation for pre- and post-ion/ion reaction data. As a rule, a significant decrease in peak heights is observed after ions are reduced in charge. In the case of Figure 2, there is a decrease in ion intensities of roughly a factor of 10 in going from the pre-ion/ion reaction spectrum to the post-ion/ion reaction spectrum. Differences in peak heights between pre- and post-ion/ion reaction spectra can arise from several factors: such as scan rate differences, neutralization, a dependence of detector gain on ion charge, and any other ion loss processes. Differences in scan rate can be accounted for by considering peak areas rather than peak heights. When the areas under the peaks for the spectra of Figure 2 were determined, it was found that there was roughly a factor of 3 loss in signal in going from the pre- to post-ion/ion reaction data. Such a loss in signal cannot be accounted for by total neutralization of some of the ions. Based on the degree of conversion of doubly charged ions to singly charged ions observed in the comparison of Figure 2, it is unlikely that more than 10% or so of the initial ion population was neutralized during the reaction period leading 3538 Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

to Figure 2b.29 Most of the loss in signal must be accounted for as being due to decreasing detector gain with decreasing charge, to ion loss from sources other than neutralization, such as ion scattering, or to a combination of the two. For fixed electric field conditions for accelerating the product ions, ion velocity is related to the square root of the ion charge and detector efficiency is related to ion velocity.56 A dependence of detector gain on ion charge might therefore be expected to account for at least some of the observed loss in signal. Unfortunately, without quantitative data on the dependence of detector gain on ion charge under the conditions used in this study, no firm conclusions regarding the extent of ion loss other than that due to total neutralization can be drawn. Ubiquitin. Ubiquitin constitutes another system for which several laboratories have reported product ion spectra for its multiply protonated parent ions formed via electrospray. A variety of activation methods have been applied to the ions and data have been reported both using a triple-quadrupole tandem mass spectrometer12,13 and FTICR instruments.54,57-59 In the latter studies, data from both collisional activation57,58 and infrared photodissociation54,59 methods have been reported. As might be expected, there are a number of differences observed in comparing the literature data for a given parent ion charge state of ubiquitin and they can arise from differences in the energies and time frames associated with the activation methods and differences in instrument discrimination effects. However, in all cases, most of the dissociation is dominated by relatively few reaction channels and the identities of many of the major products are held in common among the various studies. In this work, we add ion trap collisional activation as yet another method used to dissociate multiply charged ubiquitin parent ions. Like some of the collisional activation techniques and the infrared photodissociation methods used in the FTICR studies,54,57-59 ion trap collisional activation is a relatively slow activation method42 and is expected to yield comparable, though not necessarily identical, results. Product ion spectra have been acquired for the +12 to +4 charge states of ubiquitin. To obtain sufficient signal levels for the +6 to +4 charge states, an ion/ion reaction period following positive ion accumulation and prior to isolation of the parent ion of interest was employed. In experiments yielding post-ion/ion reaction product ion spectra for the +6 to +4 charge states, therefore, two ion/ion reaction periods were employed. The first was used to form the parent ion of interest from the parent ions of higher charge, and the second was used to convert the product ion mixture to largely singly charged ions. Figure 3 shows the pre-ion/ion reaction product ion spectra of the +11 to +8 chargestate parent ions. These data are shown primarily to illustrate changes in relative product ion signals that occur as a result of ion/ion reactions (see below). Virtually all of the peaks observed in the pre-ion/ion product ion spectra could be attributed to sequence-related ions, with benefit of the interpreted post-ion/ ion reaction product ion spectra. Only the most abundant product (56) Geno, P. W.; McFarlane, R. D. Int. J. Mass Spectrom. Ion Processes 1989, 92, 195-210. (57) Loo, J. A.; Quinn, J. P.; Ryu, S. I.; Henry, K. D.; Senko, M. W.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 286-289. (58) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012808. (59) Jockusch, J. A.; Schnier, P. D.; Price, W. D.; Strittmatter, E. F.; Demirev, P. A.; Williams, E. R. Anal. Chem. 1997, 69, 1119-1126.

Figure 3. Pre-ion/ion reaction product ion spectra of bovine ubiquitin: (a) (M + 11H)11+; (b) (M + 10H)10+; (c) (M + 9H)9+; (d) (M + 8H)8+.

ions are labeled in Figure 3 for clarity. The data of Figure 3 show significant variation in the mass/charge ratios and relative abundances of the product ions derived from parent ions of different charge. Some of the variation arises from different numbers of charges associated with common dissociation products, but much of it arises from different levels of contribution from the various competitive reaction channels. The post-ion/ ion reaction spectra described below make this clear. Figure 4 shows the post-ion/ion reaction product ion spectra from the +12 to +4 charge states of ubiquitin. Several important observations can be made from the comparison of the pre-ion/ ion reaction product ion spectra (Figure 3a-d) and the corresponding post-ion/ion reaction product ion spectra (Figure 4be). First, it is clear that the relative abundances of the product ions observed in the pre-ion/ion reaction spectra are not reproduced in the post-ion/ion reaction spectra. Ions of lower charge in the pre-ion/ion reaction data increase in abundance relative to the ions of higher charge. For example, whereas the y588+ and y589+ product ions are the most abundant ions in the pre-ion/ion reaction product ion spectrum of the (M + 11H)11+ parent ion (Figure 3a), the b18+ ion is the most abundant product ion in the post-ion/ion product ion spectrum (Figure 4b). The latter ion appeared as a triply charged ion in the pre-ion/ion product ion spectrum. Likewise, the signals arising from the y58 and b52 fragments in the pre-ion/ion data from the (M + 10H)10+ parent ion (Figure 3b) are more abundant than that of the y24 fragment, whereas the opposite is true in the post-ion/ion reaction data (Figure 4c). The y58 fragment is observed as +7 and +8 ions

and the b52 fragment appears as a +6 ion in the pre-ion/ion reaction data, whereas the y24 ion is triply charged prior to ion/ ion reaction. Many other examples can be given as it is a general observation that highly charged ions in the pre-ion/ion reaction product ion data appear at lower relative abundance in the postion/ion reaction spectra. Several effects might be considered to account for this observation. For example, ion/ion reaction kinetics might give rise to changes in relative signal levels for the various products through different degrees of total neutralization. However, this effect would predict a greater degree of total neutralization of the product ions initially formed with lower charge. It does not explain less relative signal from ions initially formed at higher charge states. In any case, different degrees of total neutralization as a function of initial ion charge are not expected to be a major source of discrimination.29,30 Some other effect that would lead to preferential loss of the ions initially present at high charge might be considered. Such an effect cannot be precluded based on these data. However, before it can be concluded that an ion loss mechanism that is ion charge dependent plays a role, the currently unquantified dependence of detector gain on ion charge, as alluded to in the discussion of Figure 2, must be determined. A second point that comes from the comparison of Figure 3 with Figure 4b-e is that the spectra in the latter figure are far more readily interpreted. In fact, many of the small signals that appear in the post-ion/ion reaction spectra can be readily identified as products that arise from sequence-informative fragmentation. The much greater charge-state overlap in the pre-ion/ion reaction Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

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Figure 4. Post-ion/ion product ion spectra of bovine ubiquitin: (a) (M + 12H)12+; (b) (M + 11H)11+; (c) (M + 10H)10+; (d) (M + 9H)9+; (e) (M + 8H)8+; (f) (M + 7H)7+; (g) (M + 6H)6+; (h) (M + 5H)5+; (i) (M + 4H)4+.

spectra, on the other hand, leads to much greater uncertainty in assigning peaks of relatively low signal-to-noise ratio. Even with the benefit of the post-ion/ion reaction spectra, some of the major peaks in Figure 3 are difficult to assign without ambiguity. For example, the peak labeled b394+;b586+ in Figure 3c cannot be assigned as arising from either one or the other product, or from a combination of both. These two ions differ in m/z by 0.4 unit and require a resolving power of about 2600 to separate them. Resolving powers of better than 4300 and 6400 are needed to resolve the isotope peaks of the b39 and b58 fragments, respectively. The post-ion/ion reaction spectrum (Figure 4d), on the other hand, clearly shows that both fragments are formed without requiring high resolving power and without charge-state ambiguity. If a detection scheme is sensitive to product ion charge, clearly the relative abundances of the ions that appear in the product ion spectrum from a multiply charged parent ion must be considered with care in drawing conclusions about the relative importance of competing reaction channels. For this reason, the post-ion/

ion reaction product ion spectra might provide a more accurate picture of the changes in dissociation behavior of multiply charged ions than do the pre-ion/ion reaction spectra. The spectra of Figure 4 reflect the dissociation behavior of a wide range of ubiquitin charge states obtained using essentially identical ion activation conditions. Furthermore, all of the ions were subjected to identical ion/ion reaction and mass analysis conditions. By converting the product ions to largely singly charged ions, the spectra of Figure 4 provide a much more readily interpreted picture of the role of parent ion charge in the dissociation behavior of ubiquitin than do the spectra of Figure 3 (and the pre-ion/ion reaction product ion spectra of the +12 and +7 to +4 charge states not shown here). Multiply charged ubiquitin parent ions fragment primarily through five major dissociation channels under ion trap collisional activation conditions, in qualitative agreement with the FTICR studies.54,57-59 The previously noted propensity for cleavage at the C-terminal side of acidic residues,60-62 aspartic acid in particular, is clearly apparent in these data. The cleavage sites Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

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of the major dissociation channels along with the complementary products formed therefrom follow: Glu18-Pro19 (y58/b18); Asp32Lys33 (y44/b32); Asp39-Gln40 (y37/b39); Asp52-Gly53 (y24/b52); and Asp58-Tyr59 (y18/b58). Another commonly observed cleavage occurs at Glu51-Asp52 (y25/b51), and a variety of other sequenceinformative fragments are observed at relatively low abundance for at least some of the parent ion charge states. The only major dissociation channel that is not adjacent to an aspartic acid residue is the Glu18-Pro19 channel leading to the y58/b18 complementary pair. This channel dominates at the highest charge states (viz., +12 to +10) but makes a much smaller contribution at charges states below +10. Evidence for the Asp58-Tyr59 channel (y18/b58 complementary pair), on the other hand, is almost nonexistent for the +12 and +11 charge states and very small for +10. However, it is the dominant channel for charge state +9 and below. Likewise, the Asp32-Lys33 (y44/b32 complementary pair) and Asp39-Gln40 (y37/b39 complementary pair) channels make no contribution to the +12 and +11 charge state spectra but contribute to varying degrees to all of the others. As with the Glu18-Pro19 channel, the Asp52-Gly53 (y24/b52 complementary pair) channel contributes to varying degrees to all of the spectra. Interestingly, most sequence information can be derived from the +7 to +10 charge state parent ions. That is, a larger number of different sequence-informative channels contribute to these product ion spectra than contribute to either the higher or lower charge-state product ion spectra. A case in point is the spectrum for the (M + 8H)8+ parent ion (Figure 4e). In addition to showing contributions from all of the major dissociation channels already mentioned, the spectrum shows, among others, y59-y65, y40, y42y44, b14-b17, and b32-b34 ions. Significantly fewer product ions are clearly apparent in the product ion spectra of the +12, +11, +6, +5, and +4 parent ions. The activation of selected parent ion charge states is necessary to determine the parent-product ion relationships associated with each parent ion charge. However, for the purpose of maximizing sequence information, it is often desirable to activate a range of parent ion charge states simultaneously and to acquire the product ion spectrum that results from the dissociation of as many parent ions as possible, regardless of initial charge. This is often effected by dissociation of ions in the vacuum-atmosphere interface63-65 of an electrospray ion source. This is usually referred to as nozzleskimmer dissociation because many electrospray interfaces employ a relatively large potential gradient between the ion sampling nozzle and a skimmer located downstream. Our interface does not employ a conventional skimmer but fragmentation in the interface can be induced by use of a large potential gradient between the atmospheric sampling aperture plate and the first lens in the interface. We refer to dissociation in this region as “interface-induced” dissociation and point out that it is expected (60) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 3015-3023. (61) Qin, J.; Chait, B. T. J. Am. Chem. Soc. 1995, 117, 5411-5412. (62) Summerfeld, S. G.; Whiting, A.; Gaskell, S. J. Int. J. Mass Spectrom. Ion Processes 1997, 162, 149-161. (63) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988, 2, 207-210. (64) Smith, R. D.; Loo, J. A.; Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R. J. Am. Soc. Mass Spectrom. 1990, 1, 53-65. (65) Kelleher, N. L.; Costello, C. A.; Begley, T. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1995, 6, 981-984.

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Figure 5. Post-ion/ion reaction electrospray mass spectrum of ions derived from interface-induced dissociation of bovine ubiquitin ions.

to be analogous to nozzle-skimmer dissociation effected in other electrospray interface geometries. As is also commonly observed in nozzle-skimmer dissociation data reported in the literature, dissociation induced in our interface tends to fragment preferentially the higher charge states. Figure 5 shows the post-ion/ion reaction mass spectrum acquired for bovine ubiquitin using a voltage difference of 230 V between the atmospheric sampling aperture and the first lens in the interface (a voltage difference of 20-30 V was used to acquire all MS/MS data). Ion/ion reaction conditions were used to yield largely singly charged ions. The interface-induced dissociation post-ion/ion reaction spectrum yielded more structurally informative fragmentation than the MS/ MS spectra either individually or collectively. Unlike the pre-ion/ ion reaction spectrum acquired using interface-induced dissociation (data not shown), most of which could not be interpreted reliably, the post-ion/ion reaction spectrum showed clearly identifiable product ions from somewhat over half of all of the possible dissociation channels that yield complementary b-type and y-type ions. The richer degree of structural information obtained from interface-induced dissociation than is obtained from the sum total of the structural information obtained from the MS/ MS spectra of the +12 to +4 charge states is probably attributable to the fact that the activation process in the interface is much more likely to yield products from consecutive fragmentation. Whatever the reason for the larger array of structurally informative fragments derived from interface-induced dissociation, ion/ion chemistry can facilitate their identification in direct analogy with the true MS/MS experiments described above. Tuna, Bovine, and Equine Cytochrome c. Some of the original work with the collisional activation of multiply charged protein ions was directed at cytochrome c using triple-quadrupole tandem mass spectrometry.66,67 Product ion spectra, for example, have been shown to serve as fingerprints in differentiating cytochrome c from nine different species.64 Assignment of the various product ion signals to sequence-related ions, however, was not made in the studies employing triple-quadrupole instrumenta(66) Smith, R. D., Barinaga, C. J.; Udseth, H. R. J. Phys. Chem. 1989, 93, 50195022. (67) Smith, R. D.; Barinaga, C. J. Rapid Commun. Mass Spectrom. 1990, 4, 5457.

Figure 6. Electrospray mass spectrum of tuna cytochrome c obtained under conditions intended to induce fragmentation.

tion due to ambiguities in charge and, therefore, mass assignment. Only with benefit of the high resolving power of high magnetic field strength FTICR could the product ion spectra of multiply charged ions derived from cytochrome c be interpreted.68 Collision-induced dissociation of multiply charged cytochrome c ions, and ions of other moderate-sized proteins, has also been effected in the quadrupole ion trap but, as with the triple-quadrupole instrumentation, interpretation of the product ion spectrum was confounded by uncertainty in product ion charge.69 The use of ion/ion reactions to eliminate product ion charge-state ambiguity becomes more valuable as the mass and charge of the protein ion increases due to the increasing number of plausible mass and charge combinations that can give rise to a product ion signal that appears in the pre-ion/ion reaction spectrum. Data from cytochrome c product ions are shown here to illustrate how ion/ ion reactions can facilitate interpretation of fragmentation from moderately high mass multiply charged ions. Figure 6 shows the electrospray mass spectrum of tuna cytochrome c under interface conditions chosen to induce fragmentation. Under normal interface conditions, charge states extending up to +17 are observed and the “chemical noise” level between charge states is well below 50 of the intensity units of Figure 6. What appears to be “chemical noise” in Figure 6 is largely due to interface-induced fragmentation. Similar spectra were also acquired with horse cytochrome c and bovine cytochrome c. Making product ion assignments to the data of Figure 6 is clearly a difficult proposition. Figure 7 shows the post-ion/ ion reaction spectrum for all three forms of cytochrome c using conditions for inducing fragmentation in the interface. Reaction conditions were employed to yield spectra dominated by singly charged ions. A large majority of the prominent product ion peaks of the spectra of Figure 7 could be assigned as either y-type or b-type ions. In the case of bovine cytochrome c, for example, product ions representing cleavage of roughly half of all possible cleavages to yield y-type and b-type ions were represented in the (68) Wu, Q.; Bakhtiar, R.; Van Orden, S.; Cheng, X.; Hofstadler, S. A.; Smith, R. D. Abstracts of Papers, 208th National Meeting of the American Chemical Society, August 21, 1994, Alestr. 97-ANYL. (69) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tuscon, AZ, June 3-8, 1990; pp 1134-1135.

Figure 7. Post-ion/ion reaction spectra of ions derived from interface-induced dissociation of three forms of cytochrome c: (a) tuna; (b) bovine; (c) equine.

post-ion/ion reaction product ion spectrum. Sequence-informative fragmentation from contiguous portions of the cytochrome c variants ranging in length up to 27 residues can be identified in the data of Figure 7. Partial sequence information derived in this way can be used to search protein databases for possible protein identification. The comparison of the data of Figure 6 with that of Figure 7a demonstrates clearly the improved facility with which product ion spectra derived from high-mass multiply charged parent ions can be interpreted by simplifying the spectra via ion/ ion chemistry. Far more information regarding the three cytochrome c variants can be obtained from Figure 7 than from the Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

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pre-ion/ion reaction electrospray mass spectra employing interface dissociation. Proteins the size of cytochrome c constitute the practical upper limit of the ion trap system used in this work for the interpretation of product ion spectra. There is a tradeoff imposed by the software of the system between upper mass limit and mass accuracy. The data system digitizes at a fixed rate so that the number of data points across a peak decreases as the m/z range of the ion trap is extended using resonance ejection. For the mass/charge range used to collect the data of Figure 7 (upper m/z limit 13 000), the mass accuracy of the system was about 500 ppm. This value does not necessarily represent the achievable limit for a quadrupole ion trap as the mass analyzer, particularly one with software and hardware designed for high m/z analysis. Furthermore, the ion trap need not serve as both the ion/ion reactor and mass analyzer to take advantage of the benefits of ion/ion chemistry for simplifying product ion spectra derived from multiply charged ions. CONCLUSIONS Multiple charging of high-mass species facilitates their dissociation and can lead to a richer array of structurally informative product ions than dissociation of the singly charged parent ion. However, dissociation of a multiply charged ion leads to a mixture of product ion masses and charges that can complicate the interpretation of the product ion spectrum. An alternative to the use of high resolving power to determine product ion charge state and, as a consequence, product ion mass, is the use of ion/ion proton-transfer reactions to reduce all product ion charges predominantly to +1. In this way, product ion charge state ambiguity is largely eliminated, thereby facilitating the interpretation of the product ion spectrum. Charge-state reduction can be effected in a quadrupole ion trap in which ions of opposite polarity can be stored simultaneously. Anions derived from glow discharge ionization of perfluorocarbons such as PDCH have been shown to react with polypeptide cations exclusively by proton transfer and do not lead either to fragmentation of the positive

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ions or to adduct formation. This is significant for the present application because it implies that ion/ion chemistry adds no new components to the product ion mixture. It simply reduces charge. The nature of ion/ion reaction kinetics allows the entire mixture of product ions to be reduced largely to singly charged species using a single reaction period. The charge-squared dependence of the ion/ion reaction rates has the beneficial effect that highly charged ions are reduced to singly charged ions very quickly such that a large fraction of product ions initially present as singly charged ions remain in the ion trap. This is significant for the present application because it implies that ion/ion chemistry removes no components initially present in the mixture. The use of ion/ion chemistry to simplify interpretation of product ion spectra results in a significant loss in signal (although signal-tonoise ratio is less affected) due to decreasing detector gain with decreasing product ion charge and/or some ion loss process other than complete neutralization. Significant changes in product ion relative abundances are also observed between pre-ion/ion reaction data and post-ion/ion reaction data from the same parent ion. The simplest explanation for this observation is a dependence of detector gain on product ion charge state. This interpretation therefore suggests that conclusions regarding the relative contributions of the various competitive dissociation channels from a multiply charged peptide or protein are most reliably drawn from post-ion/ion reaction data where differences in product ion charge states are largely eliminated. ACKNOWLEDGMENT This work was supported by the National Institutes of Health under Grant R01GM45372. Oak Ridge National Laboratory is managed for the U.S. Department of Energy under Contract DEAC05-96OR22464 by Lockheed Martin Energy Research Corp.

Received for review March 11, 1998. Accepted June 23, 1998. AC9802832