Selective Disulfide Bond Cleavage in Gold(I) Cationized Polypeptide

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Selective Disulfide Bond Cleavage in Gold(I) Cationized Polypeptide Ions Formed via Gas-Phase Ion/Ion Cation Switching Harsha P. Gunawardena,† Richard A. J. O’Hair,‡ and Scott A. McLuckey*,† Department of Chemistry, 560 Oval Drive, Purdue University, West Lafayette, Indiana 47907-2084, School of Chemistry, The University of Melbourne, Melbourne, Victoria 3010, Australia, and Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Melbourne, Victoria 3010, Australia Received June 9, 2006

Abstract: Gaseous multiply protonated disulfide-linked peptides have been subjected to reactions with AuCl2- ions to explore the possibility of effecting cation switching of Au+ for two protons and to determine whether cationization by Au+ ions affords selective dissociation of disulfide linkages. The incorporation of Au+ into several model disulfide-linked peptides proved to be straightforward. The primary ion/ion reaction channels were proton transfer, which does not lead to Au+ incorporation, and attachment of AuCl2- ions to the polypeptide cation, which does incorporate Au+. Fragmentation of the attachment product, the extent of which varied with peptide and charge state, led to losses of one or more molecules of HCl and, to some extent, cleavage of polypeptides at the disulfide linkage into its two constituent chains. Collisional activation of the intact metal-ion-incorporated peptides showed cleavage of the disulfide linkage to be a major, and in some cases exclusive, process. Cations with protons as the only cationizing agents showed only small contributions from cleavage of the disulfide linkage. These results indicate that Au+ incorporation into a disulfidelinked polypeptide ion is a promising way to effect selective dissocation of disulfide bonds. Cation switching via ion/ion reactions is a convenient means for incorporating gold and is attractive because it avoids the requirement of adding metal salts to the analyte solution. Keywords: disulfide-linked peptides • ion/ion chemistry • gold(I) cationization • tandem mass spectrometry

Introduction The utility of tandem mass spectrometry in proteomics is dependent upon the structural information derived from dissociation of a gas-phase ion that serves as a surrogate for a peptide or protein of interest. The dissociation chemistry of a peptide or protein ion is highly dependent upon its charge state, polarity, and nature of the ion (e.g., cation or anion * To whom correspondence should be addressed. E-mail: mcluckey@ urdue.edu. Phone: (765) 494-5270. Fax: (765) 494-0238. † Purdue University. ‡ School of Chemistry, The University of Melbourne, and Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne. 10.1021/pr0602794 CCC: $33.50

 2006 American Chemical Society

radical, protonated or deprotonated molecule, metal cationized species, etc.). It is often the case that different ion types derived from the same neutral precursor peptide provide complementary structural information when they dissociate. This is often the case, for example, when protonated or multiply protonated peptides are subjected to collision-induced dissociation (CID)1 versus when they are first converted to hypervalent cation radicals following the capture of a free electron or transfer of an electron from a negative ion. The latter processes are referred to as electron capture dissociation (ECD)2,3 and electron-transfer dissociation (ETD),4 respectively, and they tend to cleave polypeptides at the N-CR bond of the peptide backbone, whereas CID of protonated species tends to cleave at amide linkages. The number and identities of dominant cleavage channels often gives rise to differences in the extent of sequence information available from the dissociation products. The dissociation behavior of modified peptides and proteins is also highly dependent upon the nature of the gasphase ion to which they are converted. In the case of CID versus ECD or ETD of multiply protonated peptides, quite distinct differences in the dissociation behaviors of modified peptide and protein ions are observed. For example, post-translational modification that leads to relatively labile substituents, such as phosphorylation and glycosylation, often lead to the loss of the substituent under CID conditions;5 ECD and ETD, on the other hand, tend to cleave the polypeptide backbone without loss of the substituent.6-8 The formation of disulfide linkages in proteins is an important post-translational process leading to the stabilization of protein structures.9 Disulfide linkages when present in gaseous peptide and protein ions can have a major influence on the dissociation chemistry of the ions. In the case of multiply protonated peptides or proteins under commonly used CID conditions, backbone cleavages often dominate the fragmentation chemistry with little or no apparent evidence for cleavage of the disulfide linkage. However, the disulfide linkages can stabilize regions of the ion that fall within loops formed by the disulfide bond thereby affecting the extent of primary structural information inherent in the product ions.10 It has been shown, however, that when proton mobility within the ion is limited, disulfide bond cleavage can be dominant in the CID of cations11 and is ordinarily dominant in the CID of multiply charged anions.12 Recently, protein disulfide bonds have been mapped using broad band CID of multiply charged peptide anions.13 It has also been shown that ECD and ETD tend to favor disulfide bond cleavage,14,15 although backbone cleavages can be quite Journal of Proteome Research 2006, 5, 2087-2092

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communications Table 1. Peptide Sequences and Designations Used in Text

competitive at high charge states. Facile disulfide bond cleavage in peptides via ultraviolet photodissociation (UVPD) has also been reported.16 In contrast with ECD and ETD, both interand intramolecular disulfide bonds can be cleaved without reducing the parent ion charge state via UVPD. A possible alternative approach for selective disulfide bond cleavage is to form metal-cationized ions using metals with relatively high affinities for association with sulfur. Reactions of transition metal ions, such as Fe+ and Cu+, are known to cleave bonds in organic molecules in the gas phase,17 and cleavage of disulfide bonds, in particular, has been noted for Fe- and Co-.18 Evidence for cleavage of disulfide bonds in the gas phase was presented in an ion/ion reaction study involving the reaction of Fe+ with multiply charged anions for insulin,19 although it is not clear if the fragmentation was due to selective Fe+ chemistry or excitation of the insulin anions (which also cleave at disulfide bonds under CID conditions12). In this work, we have specifically examined Au+ for its potential for selective disulfide bond cleavage in polypeptide cations. Gold(I) is known to have a particularly high affinity for sulfur-containing ligands both in the gas phase20 and condensed phase,21 and there are examples of gold(I) complexes facilitating disulfide bond cleavage in biologically relevant systems.22,23 It is therefore reasonable to expect that gas-phase cationization of disulfidecontaining polypeptides might lead to association of the cation with the disulfide linkage with the resulting facilitation of cleavage at that site. Overall, this work is motivated by two major objectives. First, it is of interest to develop means for selective and complementary dissociation chemistries of modified peptides so as to maximize structural information, with the focus here being selective cleavage of disulfide bonds. The application can be for use in disulfide mapping strategies or for the gas-phase cleavage of disulfide bonds to enable subsequent structural interrogation via CID. An alternative means to this end might be the formation of metal-cationized polypeptides via the mixing of the peptide with metal-containing salts in the electrospray solution, which has also been shown to be effective for peptides with disulfide linkages.24 The second major objective is to form the metal-cationized ion in the gas phase from, in this case, a multiply protonated version of the ion via ion/ ion reactions. Such a strategy has already been demonstrated for a number of metal ions with both peptides and proteins.25,26 The successful achievement of this second objective would allow for the avoidance of both condensed-phase chemistries for disulfide bond reduction and the need for the inclusion of metal salts in the electrospray solution with the concomitant increase in mass spectral complexity. 2088

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Experimental Section Acetonitrile, glacial acetic acid, hydrochloric acid, and trifluoroacetic acid (TFA) were purchased from Mallinckrodt (Phillipsburg, NJ). The peptides somatostatin 1-14 and Argconopressin G were purchased from Bachem (King of Prussia, PA). TPCK-treated trypsin, ammonium bicarbonate, and gold(III) chloride were purchased from Sigma-Aldrich (St. Louis, MO). Peptides were digested using TPCK-treated trypsin with a 1:100 enzyme/peptide ratio in 20 mM ammonium bicarbonate buffered at pH ∼ 8 from 15 min up to 2 h to obtain a partially or completely digested peptide sequence. The three tryptic peptides are listed as peptide-I, II, and III and their sequences are listed in Table 1. Peptide digestion was terminated by freezing the sample for a few minutes followed by reversed-phase separation on an Agilent (Santa Clara, CA) model 1100 HPLC, using a Poros (Applied Biosystems, Foster City, CA) R1/10 100 mm × 2.1 mm i.d. column operated at 0.5 mL/min. A linear 20-min gradient from 0 to 100% B was used, where buffer A was 0.1% aqueous TFA and buffer B was 60% acetonitrile/40% H2O containing 0.09% aqueous TFA. The collected fractions were evaporated to dryness in a speedvac and redissolved in 100 µL of solvent mixture of acetonitrile/ water/acetic acid (49.5/49.5/1 (v/v/v)) from which they were ionized via positive nano-electrospray. Gold(I) chloride anions were introduced via negative nano-electrospray of an aqueous solution of approximately 16 mM AuCl3 and 0.1 M HCl. Under these conditions, two major anion types, AuCl4- and AuCl2-, dominated the negative ion spectrum with the latter being most abundant. Most experiments were performed using a modified Finnigan Ion Trap Mass Spectrometer (ITMS) equipped with four ion sources (two electrospray ionization sources and two atmospheric sampling glow discharge ionization sources) as described in detail elsewhere.27 In brief, a DC turning quadrupole allows sequential injection of ions from three ion sources through an ion trap end-cap electrode. The fourth source and associated ion optics directly inject ions through a hole in the ion trap ring electrode. The injection and timing of all sources are controlled by the ITMS software. In this report, all experiments were performed using two independent electrospray ionization sources on the front end of the DC turning quadrupole to generate both positive and negative ions. Nanoelectrospray was performed using borosilicate glass capillaries (0.86 mm i.d., 1.5 mm o.d.) that were pulled using a P-87 Flaming/Brown micropipet puller (Sutter Instruments, Novato, CA) to form nano-electrospray emitters. A stainless steel wire, attached to an electrode holder (Warner Instruments, Hamden, CT), was inserted into the capillary, and a potential of 1-2 kV was applied to the wire to induce electrospray. Peptide cations were first accumulated, and the charge state of interest was

communications Scheme 1. Reaction Scheme for Doubly Protonated DisulfideLinked Peptide, M, with AuCl2-

isolated in the ion trap, followed by injection and isolation of the gold chloride anions. The peptide cations and gold anions were stored together in the ion trap over a variable reaction period to effect ion/ion reactions. Selected product ions were subjected to collision induced dissociation (CID) with an appropriate ion activation voltage. Ion isolation steps were performed by rf ion isolation ramps tuned to eject ions from selected ranges of mass-to-charge ratio.28 In the case of the isolation of the negative reagent ion, it was not always possible to eject all of the unwanted anions without affecting the population of cations already isolated in the ion trap. Therefore, small amounts of AuCl4- ions were often present during the ion/ion reaction period. Mass analysis was performed via resonance ejection.29 An ion/ion reaction experiment involving doubly protonated Peptide I and AuCl2- ions was conducted using a quadrupole/ time-of-flight tandem mass spectrometer to provide better mass measurement accuracy than that available with the ion trap instrument mentioned above. The experiment was conducted to verify product ion assignments made herein. This instrument, its modifications, and the procedure for conducting ion/ion reactions have recently been described.30

Results and Discussion The ion/ion reaction method for incorporation of metal cations into multiply protonated polypeptides has been termed “cation switching” and involves the replacement of (n + 1) protons with a metal cation, where n is the oxidation state of the metal. The metal ion is delivered as a negatively charged complex by forming complexes with the net charge of the anionic ligands exceeding that of the metal ion. For the incorporation of Au(I) ions in this study, chloride was used as the anionic ligand. Scheme 1 shows a simplified scheme for the origin of the ion/ion reaction products typically observed in a cation switching experiment involving, in this case, two peptide chains bound by a disulfide linkage, represented as M, carrying two positive charges in reaction with AuCl2-. The scheme shows the initial formation of an electrostatically associated complex, indicated with a rate constant, kpair, the formation of which is believed to be rate-limiting in gas-phase ion/ion reactions of large polyatomic systems.31 From this interaction, two major processes compete. The two reactants can come into close enough proximity to form a long-lived

chemical complex, indicated by the rate constant kc, or a charge transfer at a crossing point can occur without formation of a long-lived chemical complex, indicated by kH+-hop. (Note that, in principle, electron transfer at a crossing point can also occur, but no evidence for electron transfer was noted in this work.) The formation of an intermediate excited chemical complex, represented in the scheme as [(M+2H- -AuCl2-]+*, is expected to be a necessary step in the incorporation of Au+ into the ion. The excited intermediate complex can be stabilized by collisions or light emission, or it can dissociate by loss of HCl, AuCl, HCl + AuCl, or 2HCl. The loss of two molecules of HCl gives rise to the [M + Au]+ species of interest, which may be observed as a stable product or may dissociate further. Scheme 1, or analogous ones for other peptide ion charge states, is useful to consider in interpreting the spectra resulting from the ion/ion reaction. Figure 1 shows the major products formed in the ion/ion reaction of the doubly charged version of Peptide I with AuCl2- (note: small amounts of AuCl4- were also present during the reaction period). The vertical scale is expanded by 3× to show some of the products of relatively low abundance. The [M + H + AuCl]+/[M + H]+ abundance ratio is roughly 7.7 in this case, indicating that proton transfer is only a very minor reaction channel with this set of reactants. The major products are the singly protonated ion, which could arise from the proton hopping mechanism or from the losses of HCl and AuCl from the attachment of the anion to the cation. However, collisional activation of the [M + H + AuCl]+ species (see below) showed no evidence for AuCl loss. Hence, the [M + H]+ ion likely arises via the proton hopping mechanism. Evidence for the attachment of AuCl2- and AuCl4-, the latter to a much lesser degree due to its much lower abundance in the anion population, is also apparent. The most abundant products, however, appear to arise from loss of HCl or 2HCl from the AuCl2- adduct to yield the [M + H + AuCl]+ and [M + Au]+ ions, respectively. It is also of interest that small signals corresponding to each of the peptide chains also appear, which suggests the possibility that some of the [M + Au]+ ions fragment further by cleaving between the S-S bond of the disulfide linkage, although other routes to the peptide chains cannot be precluded. The possibility for sequential fragmentation as the origin for the ionized peptide chains is supported by experiments involving the CID of the [M + H + AuCl]+ and [M + Au]+ ions. Figure 2a shows the CID results for the [M + H + AuCl]+ parent ion, which shows a dominant loss of HCl to give the first generation product [M + Au]+ as well as A-chain and B-chain products, which are expected to arise either largely or exclusively from dissociation of the [M + Au]+ ion. Figure 2b shows the CID spectrum of the [M + H]+ ion, which shows that disulfide bond cleavage contributes relatively little to the dissociation of the ion under these conditions. The product ion symbols in the CID spectrum indicate the chain from which a particular product ion is formed by showing the intact chain first, followed by the chain from which a fragment arises, followed in parentheses by the conventional symbol for the fragment from a single chain. For example, the designation AB(b3) indicates a b3-type fragment from the B-chain, which remains attached to the intact A-chain. In the case of Peptide I, the AB(b3) and BA(b3) ions are isomeric. Ion/ion reactions were performed in a modified quadrupole/ time-of-flight tandem mass spectrometer to obtain accurate mass measurements. These experiments yielded masses (accuracy < 20 ppm) consistent with products of the type indicated in Scheme 2. That is, the major signals associated Journal of Proteome Research • Vol. 5, No. 9, 2006 2089

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Figure 1. Post-ion/ion reaction mass spectrum involving the [M + 2H]2+ ion of peptide I and AuCl2- (with a small contribution from reactions with AuCl4-).

Figure 2. CID spectra of (a) the [M + H + AuCl]+ and (b) [M + H]+ ions derived from Peptide I. Scheme 2. Generalized Scheme for the Major Dissociation Products Observed for the Cleavage of Disulfide-Linked Polypeptide [M + Au]+ Ions

with each chain correspond to a protonated version of indicated peptide sequence (i.e., with a normal cysteine residue). The mechanistic details of the reaction have yet to be investigated in detail. However, it is apparent that the gold cation facilitates cleavage of the S-S bond of the disulfide linkage much more than the weaker C-S bonds.16 (Only very small signals corresponding to cleavages at C-S bonds were noted.) When CID of protonated11 or deprotonated peptides12 gives rise to disulfide bond cleavages, the C-S bonds tend to be more readily dissociated than the S-S bond. When triply protonated Peptide I species were subjected to ion/ion reactions with AuCl2- ions (data not shown), the major first generation ion/ion reaction products were the [M + 2H]2+ ion (formed via proton transfer), the [M + 2H + AuCl]2+ ion (presumably formed via AuCl2- attachment and subsequent HCl loss), and the [M + H + Au]2+ ion (formed via AuCl2attachment and loss of 2HCl). No clear evidence for the intact attachment product, [M + 3H + AuCl2]2+, was noted, which may reflect the greater exothermicity associated with the +3/-1 reaction than for the +2/-1 reaction. Greater evidence for A-chain and B-chain ions was also noted. 2090

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Figure 3. MS/MS spectra of the Peptide I [M + H + Au]2+ (a) and [M + 2H]2+ (b) ions.

It is of interest to examine the dissociation behavior of doubly charged disulfide-linked peptide ions in which one of the excess charges is a gold ion and the other is a proton. Figure 3 compares the ion trap CID results for the relatively high amplitude activation of the [M + 2H + AuCl]2+ ion of Peptide I (Figure 3a) with the CID data for the corresponding [M + 2H]2+ ion (Figure 3b). As expected from the use of a relatively high activation amplitude, the [M + 2H + AuCl]2+ ion readily loses HCl, and the resulting [M + H + Au]2+ ion fragments further. These second generation products can be compared directly to the results obtained for activation of the [M + 2H]2+ ions. The doubly protonated ions show essentially no evidence for disulfide bond cleavage, whereas most of the product ion signal from the Au+-containing dication results from disulfide bond cleavage between the sulfur atoms. It is noteworthy that the gold atom appears in some of the product ions in the data for the doubly charged ions, whereas it did not in the singly charged ion case (Figure 2a). This is a result of the fact that there is an excess proton that can provide charge to the goldcontaining fragments (see Scheme 2). Both possible complementary pairs (i.e., [NCPR + Au]+/[CFIR + H]+ and [NCPR + H]+/[CFIR + Au]+) are observed indicating that there does not appear to be a strong preference for the gold atom to be associated with either of the chains, which is also consistent with the data for the singly charged ions. The results for Peptide I illustrate much of the overall phenomenology associated with the reactions of AuCl2- with disulfide-linked polypeptides studied to date. However, the two chains of Peptide I are of similar size and both contain a

communications -.

Figure 4. Post-ion/ion reaction spectra obtained for the [M + 3H]3+ ions of (a) Peptide II and (b) Peptide III in reaction with AuCl2-.

C-terminal arginine residue. Perhaps for this reason, the partitioning of the gold ion between the two chains upon dissociation does not show a dramatic preference for either chain. However, this is not a universal observation, as illustrated with data obtained from peptides II and III. Figure 4 compares the post-ion/ion reaction spectra of the (M + 3H)+/ AuCl2- reactions for peptides II (Figure 4a) and III (Figure 4b). In the case of Peptide II, there is a strong preference for the formation of the complementary pair [TFTSC + H]+/[AGCK + Au]+ upon dissociation of the [M + H + Au]2+ ion. Minimal evidence for the formation of the [TFTSC + Au]+/[AGCK + H]+ complementary pair is present in Figure 4a. Peptides II and III share the same Chain B and differ in Chain A by the additional NFFWK residues in Peptide III at the C-terminal side. The ion/ ion reaction behaviors of the [M + 3H]3+ ions of Peptides II and III are quite distinct. The Peptide III reaction apparently leads to complete dissociation of the AuCl2- attachment product because there is no evidence for [M + 3H + AuCl2-]2+, [M + 2H + AuCl-]2+, or [M + H + Au]2+ ions. However, evidence for the transient formation of these ions is apparent in the appearance of the [TFTSC + Au]+/[AGCKNFFWK + H]+ complementary pair. No evidence for the formation of the other possible complementary pair, [TFTSC + H]+/[AGCKNFFWK + Au]+, is apparent in the spectrum. Some evidence for gold association with Chain A of Peptide III is apparent, however, in the appearance of the [AGCKNFFWK + H + Au]2+ ion. This fragment is presumably formed by losses of 2HCl and the neutral Chain B from the [M + 3H + AuCl2-]2+ ion. This stands in contrast with the data for Peptide II in which the gold ion tends to associate with Chain A rather than Chain B. These results illustrate that highly selective partitioning of the gold ion between sides of the disulfide bond can occur. More extensive studies are necessary to shed light on the factors that affect this partitioning. Nevertheless, all disulfide-linked peptides studied to date show cleavage at the disulfide linkage when Au+ is incorporated into the ion.

Conclusions Negative nano-electrospray of an aqueous acid solution of AuCl3 yields abundant AuCl2- ions, in addition to AuCl4- ions. Singly charged gold ions can be incorporated into multiply protonated polypeptides via ion/ion cation switching with

AuCl2 When two peptide chains are linked via a disulfide bond, cations into which Au+ has been incorporated cleave predominantly at the sulfur-sulfur bond. In some cases, the cleavage occurs directly as a result of the ion/ion reaction. For those ions that do not show fragmentation of the peptide ion directly from the ion/ion reaction, subsequent collisional activation of Au+-containing ions results in a significant degree of disulfide bond cleavage. In the peptides studied here, only minor contributions, at most, from disulfide linkage cleavage was observed upon collisional activation of singly or multiply protonated species. These results suggest that Au+ cationization of disulfide-linked peptides can lead to selective dissociation of disulfide bonds and that cation switching is an effective means for incorporating Au+ into multiply protonated peptides in the gas phase. Further studies will be directed to the apparent selectivity associated with gold ion partitioning between sides of the cleaved disulfide bond and the utility of Au+ incorporation via cation switching into disulfide-linked protein ions.

Acknowledgment. This research was sponsored by the National Institutes of Health, Institute of General Medical Sciences under Grant GM 45372 and by the Division of Chemical Sciences, and Biosciences, Office of Basic Energy Sciences, U.S. Department of Energy, under Award No. DEFG02-00ER15105. R.A.J.O. thanks the Faculty of Science at the University of Melbourne for teaching release and financial support for a sabbatical through the Special Studies Program (Long). David E. Erickson is acknowledged for assistance with acquisition of the high mass accuracy data. References (1) Wells, J. M.; McLuckey, S. A. Methods Enzymol. 2005, 402, 148185. (2) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (3) Zubarev, R. A. Mass Spectrom. Rev. 2003, 22, 57-77. (4) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528-9533. (5) Flora, J. W.; Muddiman, D. C. Anal. Chem. 2001, 73, 3305-3331. (6) Stone, D. H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19-22. (7) Coon, J. J.; Syka, J. E. P.; Shabanowitz, J.; Hunt, D. F. Int. J. Mass Spectrom. 2004, 236, 33-42. (8) Hogan, J. M.; Pitteri, S. J.; Chrisman, P. A.; McLuckey, S. A. J. Proteome Res. 2005, 4, 628-632. (9) Thornton, J. M. J. Mol. Biol. 1981, 151, 261-287. (10) Stephenson, J. L., Jr.; Cargile, B. J.; McLuckey, S. A. Rapid Commun. Mass Spectrom. 1999, 13, 2040-2048. (11) Wells, J. M.; Stephenson, J. L., Jr.; McLuckey, S. A. Int. J. Mass Spectrom. 2000, 203, A1-A9. (12) Chrisman, P. A.; McLuckey, S. A. J. Proteome Res. 2002, 1, 549557. (13) Zhang, M.; Kaltashov, I. A. Anal. Chem. 2006, 78, 4820-4829. (14) 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, 2857-2862. (15) Chrisman, P. A.; Pitteri, S. J.; Hogan, J. M.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2005, 16, 1020-1030. (16) Fung, E. M. Y.; Kjeldsen, F.; Silvra, O. A.; Chan, D. T. W.; Zubarev, R. A. Angew. Chem., Int. Ed. 2005, 44, 6399-6403. (17) Eller, K.; Schwarz, H. Chem. Rev. 1991, 91, 1121-1177. (18) Sallans, L.; Lane, K. R.; Freiser, B. S. J. Am. Chem. Soc. 1989, 111, 865-873. (19) Payne, A. H.; Glish, G. L. Int. J. Mass Spectrom. 2001, 204, 47-54. (20) Schro¨der, D.; Schwarz, H.; Hrusˇa´k, J.; Pyykko¨, P. Inorg, Chem. 1998, 37, 624-632. (21) Shaw, C. F., III. Chem. Rev. 1999, 99, 2589-2600. (22) Ashraf, W.; Isab, A. A. J. Coord. Chem. 2004, 57, 337-346. (23) Deponte, M.; Urig, S.; Arscott, L. D.; Fritz-Wolf, K.; Reau, R.; Herold-Mende, C.; Koncarevic, S.; Meyer, M.; Davioud-Charvet, E.; Ballou, D. P.; Williams, C. H., Jr.; Becker, K. J. Biol. Chem. 2005, 280, 20628-20637.

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communications (24) Lioe, H.; Duan, M.; O’Hair, R. A. J. Proceedings of the 54th ASMS Conference on Mass Spectrometry, Seattle, WA, May 28-June 1, 2006. (25) Newton, K. A.; McLuckey, S. A. J. Am. Chem. Soc. 2003, 125, 12404-12405. (26) Newton, K. A.; Amunugama, R.; McLuckey, S. A. J. Phys. Chem. A 2005, 109, 3608-3616. (27) Badman, E. R.; Chrisman, P. A.; McLuckey, S. A. Anal. Chem. 2002, 74, 6237-6243. (28) McLuckey, S. A.; Goeringer, D. E.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1991, 2, 11-21.

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(29) Kaiser, R. E.; Cooks, R. G.; Stafford, G. C.; Syka, J. E. P.; Hemberger, P. H. Int. J. Mass Spectrom. Ion Processes 1991, 106, 79-115. (30) Xia, Y.; Chrisman, P. A.; Erickson, D. E.; Liu, J.; Liang, X.; Londry, F. A.; Yang, M. J.; McLuckey, S. A. Anal. Chem. 2006, 78, 41464154. (31) Wells, J. M.; Chrisman, P. A.; McLuckey, S. A. J. Am. Chem. Soc. 2003, 125, 7238-7249.

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