Ion Trap Collisional Activation of the (M + 2H)2+ − (M + 17H)17+ Ions

Anal. Chem. 2000, 72, 899-907

Ion Trap Collisional Activation of the (M + 2H)2+ (M + 17H)17+ Ions of Human Hemoglobin β-Chain T. Gregory Schaaff, Benjamin J. Cargile, James L. Stephenson, Jr.,* and Scott A. McLuckey†

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

The parent ions of human hemoglobin β-chain ranging in charge from 2+ to 17+ have been subjected to ion trap collisional activation. The highest charge-state ions (17+ to 13+) yielded series of products arising from dissociation of adjacent residues. The intermediate charge-state ions (12+ to 5+) tended to fragment preferentially at the N-terminal sides of proline residues and the C-terminal sides of acidic residues. Many, but not all, of the possible cleavages at proline, aspartic acid, and glutamic acid residues were represented in the spectra. The lowest charge-state ions were difficult to dissociate with high efficiency and yielded spectra with poorly defined product ion signals. This observation is attributed to sequential fragmentations arising from losses of small molecules such as water and/or ammonia. The poor fragmentation efficiency observed for the low charge states is due at least in part to the low trapping wells used to store the ions. Higher ion stabilities due to lower Coulombic repulsion and charges being sequestered at highly basic sites may also play an important role. Ion/ion proton-transfer reactions involving protein parent ions allows for the formation of a wide range of parent ion charge states. In addition, the ion/ion proton-transfer reactions involving protein dissociation products simplify interpretation of the product ion spectra.

The expanded role of mass spectrometry in protein analysis has been fueled in large part by advances in both ionization techniques and instrumentation. Matrix-assisted laser desorption1,2 and electrospray3,4 ionization techniques, for example, have provided means for producing gaseous peptide and protein ions. In terms of instrumentation, three dominant forms of mass analysis have emerged in biological mass spectrometry; they include those that rely on the motion of ions in electrodynamic fields (e.g., the quadrupole mass filter and quadrupole ion trap5), * Corresponding author: (phone) (423) 574-2848; (fax) (423) 576-8559; (e-mail) [email protected]. † Current address: 1393 Brown Laboratory, Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393. (1) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1202A. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (4) Gaskell, S. J. J. Mass Spectrom. 1996, 32, 677-688. (5) March, R. E. J. Mass Spectrom. 1997, 32, 351-369. 10.1021/ac991344e CCC: $19.00 Published on Web 01/26/2000

© 2000 American Chemical Society

time of flight,6 and Fourier transform ion cyclotron resonance (FTICR).7 Each form of instrumentation, as well as hybrid systems that combine different types of mass analyzers,8-10 has a unique set of characteristics that makes it competitive in at least some applications. These characteristics include mass resolution, mass measurement accuracy, speed, cost, mass-to-charge range, etc. The options available with respect to both ionization and mass analysis have enabled a wide variety of measurement strategies to be developed to address specific issues in peptide and protein science.11-14 An example of an emerging strategy, which has been pioneered by McLafferty and co-workers,15-17 involves tandem mass spectrometry applied to whole protein ions. This strategy has been enabled by the combination of electrospray ionization with high magnetic field strength FTICR. The high resolving power of high-field FTICR facilitates the interpretation of product ion spectra derived from multiply charged parents via measurement of the spacings of the isotopic peaks associated with the product ions.18 A key element to strategies that involve the tandem mass spectrometry of whole protein ions is the ability to obtain useful structural information from the ions. For this reason, McLafferty and co-workers have explored a wide range of techniques for dissociation of whole protein ions. Most of these have involved methods for activating multiply protonated proteins.19-21 However, (6) (7) (8) (9)

(10)

(11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21)

Guilhaus, M. J. Mass Spectrom. 1995, 30, 1519-1532. Amster, I. J. J. Mass Spectrom. 1996, 31, 1325-1337. Qian, M. G.; Lubman, D. M. Anal. Chem. 1995, 67, 234A-242A. Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R. S.; Hoyes, J.; Bateman, R. H. Rapid Commun. Mass Spectrom. 1996, 10, 889896. Shevchenko, A.; Chernusevich, I.; Ens, W.; Standing, K. G.; Thomson, B.; Wilm, M.; Mann, M. Rapid Commun. Mass Spectrom. 1997, 11, 10151024. Yates, J. R., III J. Mass Spectrom. 1998, 33, 1-19. Siuzdak,G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11290-11297. Yates, J. R., III Methods Enzymol. 1996, 271, 351-377. Loo, J. A. Bioconjugate Chem. 1995, 6, 644-665. 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. McLafferty, F. W. Acc. Chem. Res. 1994, 8, 379-386. Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812. Henry, K. D.; McLafferty, F. W. Org. Mass Spectrom. 1990, 25, 490-492. Chorush, R. A.; Little, D. P.; Beu, S. C.; Wood, T. D.; McLafferty, F. W. Anal. Chem. 1995, 67, 1042-1046. Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. Guan, Z. Q.; Kelleher, N. L.; O’Connor, P. B.; Aaserud, D. J.; Little, D. P.; McLafferty, F. W. Int. J. Mass Spectrom. Ion Processes 1996, 157/158, 357364.

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the use of electron capture to form odd-electron protein ions has proved to be particularly promising for deriving primary sequence information from proteins.22-25 Until recently, the tandem mass spectrometry of whole protein ions has largely been restricted to FTICR instrumentation, although the earliest studies involving tandem mass spectrometry of proteins employed triple-quadrupole instrumentation.26 Interpretation of the spectra derived from triple-quadrupole instruments was complicated by product ion charge-state ambiguities. In this paper, we relate the product ion spectra derived from quadrupole ion trap collisional activation of multiply protonated human hemoglobin β-chain over the range of charge states of 2+ to 17+. This study was enabled by the use of ion/ion proton-transfer reactions27-32 to (a) make parent ions over a much wider range of charge states than is directly accessible via electrospray and (b) to facilitate interpretation of the product ion spectra by converting all product ions largely to the 1+ charge state.32 The use of ion/ion proton-transfer reactions as means for manipulating both parent ion and product ion charge states allows for a significant increase in the ion mass that can be studied via tandem mass spectrometry using a quadrupole ion trap mass spectrometer. This development is noteworthy from the standpoint of protein analyses in that strategies based upon the direct interrogation of whole protein ions can be considered using a benchtop instrument. In analogy with the work already performed to evaluate techniques to coax structural information from protein ions in the FTICR environment,19-25 methods for dissociating protein ions in the quadrupole ion trap environment must be evaluated to optimize the extent of structural information that can be obtained with this form of instrumentation. Human hemoglobin β-chain represents a protein of obvious clinical interest,33-46 the tandem (22) Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K., McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 28572862. (23) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (24) Kruger, N. A.; Zubarev, R. A.; Horn, D. M.; McLafferty, F. W. Int. J. Mass Spectrom. 1999, 187, 787-793. (25) Kruger, N. A.; Zubarev, R. A.; Carpenter, B. K.; Kelleher, N. L.; Horn, D. M.; McLafferty, F. W. Int. J. Mass Spectrom. 1999, 183, 1-5. (26) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991, 63, 24882499. (27) McLuckey, S. A.; Stephenson, J. L., Jr. Mass Spectrom. Rev. 1998, 17, 369407. (28) Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 1996, 68, 4026-4032. (29) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Chem. Soc. 1996, 118, 73907397. (30) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1998, 9, 585-596. (31) McLuckey, S. A.; Stephenson, J. L., Jr.; Asano, K. G. Anal. Chem. 1998, 70, 1198-1202. (32) Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 1998, 70, 3533-3544. (33) Ferranti, P.; Malorni, A.; Pucci, P.; Fanali, S.; Nardi, A.; Ossicini, L. Anal. Biochem. 1991, 194, 1-8. (34) Light-Wahl, K. J.; Loo, J. A.; Edmonds, C. G.; Smith, R. D.; Witkowska, H. E.; Shackleton, C. H. L.; Wu, C. S. C. Biol. Mass Spectrom. 1993, 22, 112120. (35) Witkowska, H. E.; Bitsch, F.; Shackleton, C. H. L. Hemoglobin 1993, 17, 227-242. (36) Nakanishi, T.; Miyazaki, A.; Kishikawa, M.; Shimizu, A.; Yonezawa, T. J. Am. Soc. Mass Spectrom. 1996, 7, 1040-1049. (37) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1996, 68, 2482-2487. (38) Shackleton, C. H. L.; Witkowska, H. E. Anal. Chem. 1996, 68, 29A-33A. (39) Wu, J. T.; He, L.; Li, M. X.; Parus, S.; Lubman, D. M. J. Am. Soc. Mass Spectrom. 1997, 8, 1237-1246.

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mass spectrometry of which is not straightforward with conventional benchtop tandem mass spectrometers due to difficulties in interpreting the spectra. In this work, we explore the dissociation behavior of hemoglobin β-chain ions over a wide range of charge states using conventional ion trap single-frequency collisional activation.46 This activation technique shares many of the characteristics of the “slow-heating” methods47 employed in FTICR instrumentation such as collisional activation techniques like sustained off-resonance irradiation48 and optical methods like continuous-wave infrared multiphoton dissociation.49 While the results reported here are most directly relevant to collisional activation in the ion trap, the dissociation behavior of multiply protonated ions as a function of charge state has implications for all forms of tandem mass spectrometry. EXPERIMENTAL SECTION Human hemoglobin (HG) was purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. To obtain the desired concentration for mass spectral studies, an aqueous stock solution of typically 100-150 µM was diluted to 5-10 pmol/µL in 50:50 water/methanol with 1% acetic acid. Nanoelectrospray50 of the resulting dilute solution was performed using a patch clamp/0.9 mm borosilicate micropipet assembly (World Precision Instruments, Sarasota, FL). The potential of the stainless steel wire of the nanospray assembly was typically held at +1-1.3 kV. Flow rates ranged from 20 to 50 nL/min. The electrospray interface51 and overall instrumental setup for ion/ ion reactions52 have been described previously. The typical experimental procedure entailed isolation of ions corresponding to a specific charge state of β-chain hemoglobin. Parent ion isolation was affected with two resonance ejection ramps (30 ms rf sweep from 1150 to 7500 V0-p; 18 Vp-p sine wave applied to the end cap electrodes).53 Ions with charge states between +17 and +13 were isolated directly from the starting electrospray ion population. For the +12 to +2 charge states, the starting electrospray ion population was subjected to proton(40) Yang, L. Y.; Lee, C. S.; Hofstadler, S. A.; Pasa-Tolic, L.; Smith, R. D. Anal. Chem. 1998, 70, 3235-3241. (41) Prome, D.; Prome, J. C.; Deon, C.; Groff, P.; Kalmes, G.; Galacteros, F.; Wajcman, H. J. Mass Spectrom. 1995, (Suppl. S), S165-S168. (42) Nakanishi, T.; Miyasaki, A.; Kishikawa, M.; Shimizu, A.; Aoki, Y.; Kikuchi, M. Hemoglobin 1998, 22, 23-35. (43) Prome, D.; Deon, C.; Prome, J. C.; Wajcman, H.; Galacteros, F.; Blouquit, Y. J. Am. Soc. Mass Spectrom. 1996, 7, 163-167. (44) Frischknecht, H.; Ventruto, M.; Hess, D.; Hunziker, P.; Rosatelli, M. C.; Cao, A.; Breitenstein, U.; Fehr, J.; Tuchschmid, P. Hemoglobin 1996, 20, 31-40. (45) Shackleton, C. H. L.; Witkowska, H. E. In Mass Spectrometry: Clinical and Biomedical Applications; Desiderio, D. M., Ed.; Plenum: New York, 1994; Vol. 2, p 135. (46) 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. (47) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 32, 461-474. (48) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (49) Woodin, R. L.; Bohmse, R. S.; Beauchamp, J. L. J. Am. Chem. Soc. 1978, 100, 3248-3250. (50) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167-180. (51) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1295. (52) Stephenson, J. L., Jr.; McLuckey, S. A. Int. J. Mass Spectrom. Ion Processes 1997, 162, 89-106. (53) McLuckey, S. A.; Goeringer, D. E.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1991, 2, 11-21.

transfer reactions with anions derived from the glow discharge ionization54 of perfluoro-1,3-dimethylcyclohexane (PDCH) in order to reduce the distribution of charge states to values centered around the charge state of interest. The charge state of interest was then isolated by resonance ejection, as described above. Collisional activation of the +17 to +6 charge states was effected at a qz value of 0.2. For the +5 to +2 charge states, a qz value of 0.2 was unattainable due to the limited maximum rf output of 7500 V0-p. At the maximum rf level, qz values for the +5, +4, +3, and +2 charge states were 0.19, 0.15, 0.11, and 0.074, respectively. The amplitude of the resonance excitation voltage applied to the end caps and duration of the collisional activation period were varied to achieve the highest fragmentation efficiency for each of the ions studied. The collisional activation period ranged from 300 to 500 ms, and the amplitude applied to the end caps of the ion trap was 200 mV to 350 mV0-p. After the collisional activation period, anions were injected into the ion trap for 20 ms followed by a cation/anion mutual storage period of 100-130 ms. During this reaction period, most product ions were converted to singly charged ions. A 10-20 ms ramp of the rf amplitude was used to eject all ions below m/z 650 prior to mass analysis. This step was performed to eject the residual PDCH anions so that they would not lead to deleterious effects on mass analysis.55 Mass analysis was effected by resonance ejection56 of the ions at qz ) 0.033 42 (ejection frequency of 13.2 kHz). The spectra shown are typically the average of 700-1000 individual mass analysis scans. Mass calibration of pre-ion/ion product ion spectra was achieved by using the electrospray mass spectrum of human hemoglobin as the calibration standard. To calibrate the highmass region for post-ion/ion product ion spectra, the electrospray ions were subjected to reactions with PDCH anions for 80 ms, and the resulting (1+ to 3+) charge states of the R- and β-chain ions were used for calibration. A similar procedure was used to ensure adequate isolation of the ions of interest and that off resonance power absorption57 during the ion isolation steps did not cause fragmentation of the isolated ion population. RESULTS AND DISCUSSION An electrospray mass spectrum of HG, acquired after an ion accumulation period of 30 ms, is shown in Figure 1a. The spectrum includes charge-state distributions for both the R-chain and the β-chain. Even with an ion accumulation period of 30 ms, space charge effects are clearly apparent at the highest charge states. For example, the peak widths associated with the 19+ R-chain and β-chain ions are significantly broader that those of the 17+ ions despite the fact that the mass-to-charge (m/z) envelopes associated with the ions are narrower for the higher charge state. Such broadening is due to the well-known space charge phenomenon.58 It is most readily apparent at the highest charge states because the scan direction is from low m/z to high (54) McLuckey, S. A.; Glish, G. L.; Asano, K. G.; Grant, B. C. Anal. Chem. 1988, 60, 2220-2228. (55) Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 1997, 69, 3760-3766. (56) Kaiser, Jr., R. E.; Cooks, R. G.; Stafford, G. C., Jr.; Syka, J. E. P.; Hemberger, P. E. Int. J. Mass Spectrom. Ion Processes 1991, 106, 79-115. (57) Charles, M. J.; McLuckey, S. A.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1994, 5, 1031-1041. (58) Todd, J. F. J.; Waldren, R. M.; Freer, D. A.; Turner, R. B. Int. J. Mass Spectrom. Ion Processes 1980, 35, 107-50.

Figure 1. Electrospray mass spectra of human hemoglobin (a) before an ion/ion reaction period and (b) after an ion/ion reaction period.

m/z such that the lower charge-state ions experience less space charge because the higher charge-state ions are no longer present by the time the higher m/z ions (lower charge states) undergo resonance excitation. The space charge phenomenon also affects parent ion isolation. We found that with the ion accumulation periods necessary to generate sufficient numbers of parent ions for good product ion spectra (i.e., ∼200 ms) it was difficult to isolate the β-chain ion from the R-chain ion at charge states greater than ∼17+. Therefore, the most highly protonated species included in this study is the (M + 17H)17+ ion. For parent ions of charge less than 13+, ion/ion proton-transfer reactions were necessary to make sufficient numbers for collisional activation. Figure 1b is an electrospray mass spectrum acquired after an ion/ ion reaction period of 70 ms and illustrates the change in parent ion charge-state distribution that can be effected. The average HG ion charge state could be varied arbitrarily via selection of the ion/ion reaction time. The m/z values of the product ions formed directly via collisional activation, particularly for the high-charge-state protein ions, tend to fall within a relatively narrow range of values centered around the m/z of the parent ion. Figure 2a illustrates this scenario with the product ion spectrum obtained from the (M + 16H)16+ parent ion. The ions reflected in Figure 2a represent a complex mixture of ions ranging in mass from about 500-15 867 Da (i.e., the average mass of HG β chain) and ranging in charge from 1+ to 16+. This scenario makes interpretation of the product ion spectra particularly problematic. Figure 2b shows the product ion spectrum obtained after the ions reflected in Figure 2a were subjected to ion/ion proton-transfer reactions for 100 ms. The comparison of Figure 2 illustrates the utility of ion/ion protontransfer reactions for the assignment of product ions from dissociation of multiply charged parent ions because they can Analytical Chemistry, Vol. 72, No. 5, March 1, 2000

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Figure 2. Pre-ion/ion (a) and post-ion/ion (b) product ion spectra for the 16+ charge state of HG-β.

largely eliminate product ion charge-state ambiguity, and they can eliminate overlap of ions initially present at similar m/z values but with different masses and charges. An additional advantage to the use of ion/ion reactions in a systematic study of dissociation behavior as a function of parent ion charge is that a fixed set of mass analysis conditions can be used for every parent ion charge. Discrimination effects arising from mass analysis are therefore independent of parent ion charge such that differences in spectra can be attributed either to inherent dissociation behavior or, possibly, to differences in activation conditions. All subsequent product ion spectra discussed herein are those acquired after an ion/ion reaction period using a constant set of mass analysis conditions. A product ion spectrum for each charge state from 17+ to 2+ was collected and all are available as Supporting Information. A subset of spectra is presented here to summarize the major phenomena associated with dissociation of HG β ions. Figure 2b, which shows the post-ion/ion reaction product ion spectrum derived from the (M + 16H)16+ ion, illustrates behavior common to all of the parent ions in the charge state range of 17+ to 13+. In particular, each of the parent ions in this charge-state range exhibits series of b-type product ions corresponding to b30-b35, b37, and b39-b50. Some of the major high-mass product ions are complementary y-type products, where, for simplicity, yn′′ ions are denoted herein as yn. For example, in the case of the (M + 16H)16+ parent ion, there are product ions corresponding to y114, y112, y111, y109, y98, and y96. The latter two ions are complements of the b48 and b50 ions, respectively, while the former ions are complements of some of the b30-b37 ions. Other y-type ions that complement the b-type ions mentioned above may also be present at relatively low signal levels but cannot be clearly identified due 902

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to insufficient resolution. In any case, there is not a clear relationship between the abundances of the b-type ions and the y-type complements. While it cannot be determined from these data that sequential fragmentation plays a major role in the appearance of the product ion spectra, the relative abundances of the b30-50 ions, which are not mirrored by the relative abundances of the y-type ions, may indicate some sequential fragmentation. For example, fragmentation leading to the y111/ b35 complementary pair may result in further fragmentation of the b35 ion to the b34 ion, and so on. This possibility is suggested by the fact that the b34 ion is more abundant than the b35 ion while the y111 ion is more abundant than the y112 ion. Furthermore, a y113 ion is not clearly apparent in the spectrum, although a b33 fragment is clearly apparent. A major product ion is indicated as either the b100 or y101 ion. These product ions differ in mass by 0.1 Da and cannot be differentiated at the mass accuracy associated with these measurements. (Likewise, the respective complementary fragments y46 and b45 cannot be differentiated.) A few other small signals are also observed for this charge state but, by far, most of the fragmentation is associated with the channels leading to the y-type and b-type fragments mentioned above. The high-charge-state parent ions of the HG β-chain tend to give the most directly informative data about the primary structure of the protein due to the appearance of series of products from dissociation at adjacent residues. Chart 1 summarizes the dissociation channels observed for the 17+ to 13+ parent ions. All of the parent ions in the charge state range of 17+ to 13+ show most of the b30-b50 fragments observed with the 16+ parent. However, careful examination of the spectra shows that each parent ion behaves uniquely in terms of the relative contributions

Figure 3. Ion trap post-ion/ion reaction product ion spectrum of the HG β (M + 15H)15+ parent ion.

Chart 1

of the various fragmentation channels. An example is given in Figure 3, which shows the post-ion/ion reaction product ion spectrum of the (M + 15H)15+ parent ion. One of the major differences in this spectrum, relative to that of the (M + 16H)16+ ion (Figure 2b), is that the signal associated with the b100 or y101 fragment is significantly diminished relative to the other highmass fragment ions and signals associated with b99 and y99 fragments are apparent. The b99 signal, for example, is particularly significant because it is an abundant fragment in most of the charge states below 16+ (see below). Furthermore, the b100 or y101 fragment largely disappears from the spectra below the 15+ charge state. Another significant difference is the appearance of the y67/b79 complementary pair, which is absent in the (M + 16H)16+ parent ion data. The dissociation behavior of the 12+ to 5+ parent ions differs from the higher charge-state parents in that series of product ions from dissociation at adjacent residues, such as reflected by the b30-b50 products, are much less prevalent. Rather, the spectra tend to be dominated by a limited number of dissociation reactions from peptide linkages generally located remote from one another.

Most of the observed dissociation products arise from cleavages that might be expected on the basis of known facile fragmentations of large polypeptide and protein ions. For example, dissociations at the N-terminal side of proline residues and on the C-terminal sides of aspartic acid and, to a lesser degree, glutamic acid residues34,59-64 appear to make the major contributions to product ion spectra. There are a number of proline, aspartic acid, and glutamic residues in HG β-chain and the sites where facile fragmentation might be expected are listed in Table 1. Figure 4, which shows the post-ion/ion reaction product ion spectrum of the (M + 12H)12+ parent, is a fairly extreme example in that it shows only a single dominant dissociation with relatively little (59) (60) (61) (62)

Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1993, 65, 425-438. Qin, J.; Chait, B. T. J. Am. Chem. Soc. 1995, 117, 5411-5412. Qin, J.; Chait, B. T. Int. J. Mass Spectrom. 1999, 191, 313-320. Jockusch, J. A.; Schnier, P. D.; Price, W. D.; Strittmatter, E. F.; Demirev, P. A.; Williams, E. R. Anal. Chem. 1997, 69, 1119-1126. (63) Tsaprailis, G.; Nair, H.; Somogyi, AÄ .; Wysocki, V. H.; Zhong, W. Q.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. J. Am. Chem. Soc. 1999, 121, 51425154. (64) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012808.

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Figure 4. Ion trap post-ion/ion reaction product ion spectrum of the HG β (M + 12H)12+ parent ion.

Table 1. Possible Favored Fragmentation Sites in HG β-Chain Based on the Presence of a Proline, Aspartic Acid, or Glutamic Acid Residue potential cleavage site

complementary y/b fragments

y/b fragment masses (Da)

Thr4-Pro5 Glu6-Glu7 Glu7-Lys8 Asp21-Glu22 Glu22-Val23 Glu26-Ala27 Tyr35-Pro36 Glu43-Ser44 Asp47-Leu48 Thr50-Pro51 Asp52-Ala53 Asn57-Pro58 Asp73-Gly74 Asp79-Asn80 Glu90-Leu91 Asp94-Lys94 Asp99-Pro100 Glu101-Asn102 Glu121-Phe122 Thr123-Pro124 Pro124-Pro125

y142/b4 y140/b6 y139/b7 y125/b21 y124/b22 y120/b26 y111/b35 y103/b43 y99/b47 y96/b50 y94/b52 y89/b57 y73/b73 y67/b79 y56/b90 y52/b94 y47/b99 y45/b101 y25/b121 y23/b123 y22/b124

15417.7/451.5 15191.5/677.8 15032.4/806.9 13592.6/2276.6 13463.5/2405.7 13121.2/2748.1 12135.9/3733.3 11043.7/4825.5 10637.3/5345.1 10336.0/5533.3 10123.8/5745.5 9651.2/6218.0 7987.2/7882.0 7380.5/8488.7 6218.2/9651.0 5749.7/10119.5 5157.0/10712.2 4930.8/10938.5 2682.1/13187.2 2433.8/13435.4 2336.7/13532.6

contribution from other channels. The major dissociation arises from the cleavage at Asp99-Pro100, which, of course, is a site that satisfies two of the conditions mentioned above for facile cleavage. This dissociation is also observed in most of the higher charge states and in most of the lower charge states but in no charge state is it more dominant than for the 12+ parent ion. A more usual example of dissociation behavior associated with the intermediate charge states in shown in Figure 5, which displays the post-ion/ion reaction product ion spectrum of the (M + 7H)7+ parent ion. This spectrum shows the products from cleavage at the Asp99-Pro100 site (y47/b99) as well as products from 904 Analytical Chemistry, Vol. 72, No. 5, March 1, 2000

a number of the other sites listed in Table 1. These include products from cleavages at Glu6-Glu7 (y140), Asp21-Glu22 (y125), Glu43-Ser44 (y103/b43), Asp47-Leu48 (y99/b47), Asp52-Ala53 (y94/b52) and/or Asp94-Lys94 (y52/b94), Asn57-Pro58 (y89/b57) and/or Glu90-Leu91 (y56/b90), and Asp79-Asn80 (y67/b79). In addition to these products, there are several fragments of relatively low abundance that apparently arise from cleavages at sites other than those listed in Table 1. The most prominent set of products in this category are the y54/b92 ions that arise from cleavage at the His92-Cys93 site. It is interesting to note that, while the large majority of fragment ion signal arises from cleavages at sites listed in Table 1, less than half of the sites listed in the table are represented in the spectrum. Some, but not all, of these sites not represented in Figure 5 are observed in spectra from other parent ion charge states. A prominent example is the y111/b35 complementary pair arising from cleavage at the Tyr35-Pro36 site, which is clearly represented in the product ion spectra of the 17+ to 13+ charge states. These data suggest that in interpreting product ion spectra of a known multiply protonated protein it may be useful to identify the sites of proline, aspartic acid, and glutamic acid residues as candidate cleavage sites. However, it is clear that other factors are also important in governing protein ion dissociation and that these factors can prevent some “apparently favored” dissociations from being observed and can also promote cleavages at sites that might otherwise not be anticipated. The lowest parent ion charge states that could be induced to fragment are grouped together as the 4+ to 2+ ions. This rather arbitrary grouping is made largely on the basis of dissociation efficiency relative to ion ejection resulting from resonance excitation. Only a small percentage of parent ions (estimated at