Ion Reactions

In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics. Jong-Seo Kim , Matthew E. Monroe , David G. Camp , II , R...
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Anal. Chem. 2005, 77, 3683-3689

Sonic Spray as a Dual Polarity Ion Source for Ion/Ion Reactions Yu Xia, Xiaorong Liang, and Scott A. McLuckey*

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084

A single sonic spray source has been used to generate both positive and negative ions for subsequent ion/ion reaction experiments. Ion/ion reactions took place after ions of each polarity were sequentially injected into a linear ion trap, where axial trapping was effected by applying an auxiliary radio frequency voltage to one end lens. Absolute charge reductions via proton transfer were demonstrated for multiply charged protein/peptide cations and multiply charged oligonucleotide anions. Deprotonation of polypeptide cations occurs with anions derived from fluorinated compounds such as nonadecafluoro-1decanol and perfluoro-1-octanol, while multiply charged oligonucleotide anions are efficiently protonated via reaction with proton sponge (N,N,N′,N′-tetramethyl-1,8-naphthalenediamine) cations. No evidence for signal suppression of the biopolymer ions was noted to result from the presence of these reagents in the solution subjected to sonic spray. Several of the analytically useful applications of ion/ion proton-transfer reactions are demonstrated using a single sonic spray ion source. These include an ion parking experiment for the purpose of gas-phase ion concentration and charge-state reduction of product ions formed via beam-type and in-trap collision-induced dissociation of multiply charged oligonucleotide parent anions. Examples of complex formation are also given to illustrate the flexibility of the sonic spray-induced ion/ion reaction method. In the study of biomolecules, gas-phase ions have been served as surrogates via mass spectrometry. Ion/ion reactions in the gas phase provide the means for modifying gas-phase ions derived from biomolecules after their initial formation.1,2 This is highly desirable because the type of ion (e.g., charge state, ionizing agent, polarity) subjected to tandem mass spectrometry need not be solely determined by the ionization method. Ion/ion reactions external to a mass spectrometer have been demonstrated using either ion beams from two independent electrospray ionization sources merged at the base of a Y-tube before sampling into a quadrupole mass spectrometer3,4 or by subjecting electrospraygenerated multiply charged ions to reactions with singly charged * To whom correspondence should be addressed. Phone: (765) 494-5270. Fax: (765) 494-0239. E-mail: [email protected]. (1) McLuckey, S. A.; Stephenson, J. L., Jr. Mass Spectrom. Rev. 1998, 17, 369407. (2) Pitteri, S. J.; McLuckey, S. A. Mass Spectrom. Rev. In press. (3) Ogorzalek-Loo, R. R.; Udseth, H. R.; Smith, R. D. J. Phys. Chem. 1991, 95, 6412-6415. 10.1021/ac0481811 CCC: $30.25 Published on Web 04/19/2005

© 2005 American Chemical Society

ions formed via a radioactive polonium source5,6 or corona discharge7 coupled to a time-of-flight mass spectrometer. Strategies that employ ion/ion reactions external to the mass spectrometer enjoy the advantage of compatibility with any form of mass analyzer. However, ion manipulation capabilities are limited with these approaches. The ability to store oppositely charged ions simultaneously,8 together with ion isolation and MSn capabilities,9 makes electrodynamic ion traps flexible tools for the study and application of ion/ion reactions. Therefore, ion/ion reaction studies have been extensively performed inside an electrodynamic ion trap (either a three-dimensional ion trap or a linear ion trap), serving as both the reaction vessel and the mass analyzer.2 In all ion/ion reaction studies reported to date, two distinct sources of ions were employed to provide analyte and reagent ions, respectively. For example, the instrument geometry that has been used for many ion/ion reaction studies employs an arrangement wherein ESI-generated analyte ions are admitted to the ion trap through an end cap electrode, while an atmospheric sampling glow discharge ionization source (ASGDI)10 is used to generate reagent ions, which are injected through a hole in the ring electrode of the ion trap.11,12 The ASGDI source is most appropriate for the ionization of vapors. The reagent ions generated by ASGDI source for reducing cation charge states have commonly been anions derived from perfluorocarbons, such as perfluoro1,3-dimethylcyclohexane11,13 and perfluoro(methyldecalin).14 Multiply charged analyte anions have been subjected to reactions with cations derived from isobutylene (proton transfer15,16) and from (4) Ogorzalek-Loo, R. R.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1992, 3, 695-705. (5) Scalf, M.; Westphall, M. S.; Krause, J.; Kaufman, S. L.; Smith, L. M. Science 1999, 283, 194-197. (6) Scalf, M.; Westphall, M. S.; Smith, L. M. Anal. Chem. 2000, 72, 52-60. (7) Ebeling, D. D.; Westphall, M. S.; Scalf, M.; Smith, L. M. Anal. Chem. 2000, 72, 5158-5161. (8) Mather, R. E.; Todd, J. F. J. Int. J. Mass Spectrom. Ion Phys. 1980, 33, 159-165. (9) McLuckey, S. A.; Glish, G. L.; Van Berkel, G. J. Int. J. Mass Spectrom. Ion Processes 1991, 106, 213-235. (10) McLuckey, S. A.; Glish, G. L.; Asano, K. G.; Grant, B. C. Anal. Chem. 1988, 60, 2220-2227. (11) Stephenson, J. L., Jr.; McLuckey, S. A. Int. J. Mass Spectrom. Ion Processes 1997, 162, 89-106. (12) Reid, G. E.; Wells, J. M.; Badman, E. R.; McLuckey, S. A. Int. J. Mass Spectrom. 2003, 222, 243-258. (13) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Chem. Soc. 1996, 118, 73907397. (14) Xia, Y.; Wu, J.; Londry, F. A.; Hager, J. W.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2004, 16,71-81. (15) McLuckey, S. A.; Wu, J.; Bundy, J. L.; Stephenson, J. L., Jr.; Hurst, G. B. Anal. Chem. 2002, 74, 976-984. (16) Wu, J.; McLuckey, S. A. Int. J. Mass Spectrom. 2003, 228, 577-597.

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xenon17 or oxygen15,16 (electron transfer). The ESI/ASGDI source arrangement is generally restricted to reactions of multiply charged analyte ions with singly charged reagent ions. Instrumentation that allows for sequential injection of oppositely charged ions generated by two or three distinct ESI sources into a quadrupole ion trap via an end cap have also been described.18,19 These instruments significantly expand the range of ionic reactants amenable to study over the ESI/gas-source combinations. Recently, Hunt and co-workers reported electron-transfer ion/ion reactions in a linear ion trap (LIT), giving rise to cleavages analogous to those noted with electron capture dissociation.20 In that study, the reagent ions were generated by an additional chemical ionization source, adapted to the rear side of the modified LIT opposite from the ESI source.21 Although accommodating distinct sources provides the advantage of being able to optimize individually each ion source, it adds the complication of requiring hardware modifications. To date, the implementation of ion/ion reactions has been limited to home-built or extensively modified commercial instruments. The current work was motivated by the possibility of employing a single ionization source to form both analyte and reagent ions for the use in ion/ion reactions. In this way, such an ion source can, in principle, be added to any commercial instrument provided the potentials of the ion path can be changed to accommodate transmission of the opposite polarity ions at the appropriate times. Since at least one of the reactants must be multiply charged, a spray ionization approach is likely to be the best candidate for ionization method. Sonic spray ionization (SSI) is a spray ionization method first introduced by Hirabayashi et al. in the mid-1990s,22-24 and since then SSI has been adopted and developed by other groups.25-27 The analyte-containing solution is sprayed from a fused-silica capillary at atmospheric pressure with high-speed gas flow coaxial to the capillary. Analyte ion intensity is maximized at the sonic velocity of the gas. The origin of charged droplet formation is not fully understood, but it likely arises as a result of statistical charge distribution during droplet formation.28 A unique feature associated with SSI is that both positive and negative ions are formed simultaneously. In this study, a single sonic spray source is coupled with a hybrid triple quadrupole/LIT instrument with axial trapping induced by applying auxiliary radio frequency (rf) to an end lens of the quadrupole array. Solutions containing (17) Herron, W. J.; Goeringer, D. E.; Mcluckey, S. A. J. Am. Chem. Soc. 1995, 117, 11555-11562. (18) Badman, E. R.; Chrisman, P. A.; McLuckey, S. A. Anal. Chem. 2002, 74, 6237-6243. (19) Wells, J. M.; Chrisman, P. A.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2002, 13, 614-622. (20) Coon, J. J.; Syka, J. E. P.; Schwartz, J. C.; Shabanowitz, J.; Hunt, D. F. Int. J. Mass Spectrom. 2004, 236, 33-42. (21) 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. (22) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1994, 66, 45574559. (23) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1995, 67, 28782882. (24) Hirabayashi, A.; Hirabayashi, Y.; Sakairi, M.; Koizumi, H. Rapid Commun. Mass Spectrom. 1996, 10, 1703-1705. (25) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Anal. Chem. 2004, 76, 4050-4058. (26) Takats, Z.; Nanita, S. C.; Cooks, R. G.; Schlosser, G.; Ve´key, K. Anal. Chem. 2003, 75, 1514-1523. (27) Bjorkman, H. T.; Edlund, P. O.; Jacobsson, S. P. Anal. Chim. Acta 2002, 468, 263-274. (28) Dodd, E. E. J. Appl. Phys. 1953, 24, 73-80.

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both analyte species and components intended to give rise to oppositely charged reagent ions are subjected to SSI, and the oppositely charged ions are sequentially injected into a linear ion trap where they can react. A variety of potential reagents were examined, and those that were effective at inducing the desired ion chemistry without leading to significant analyte ion suppression in the ionization step are described here. EXPERIMENTAL SECTION A sonic spray source was built following the design of Takats et al.,26 except that an inner fused-silica capillary with internal diameter of 50 µm was used. The source was operated at a nitrogen nebulizing gas pressure of 800 kPa. Solutions containing analyte and reagent components were introduced to the SSI apparatus at a flow rate of 3 µL/min. Peptides and proteins were purchased from Sigma (St. Louis, MO) and used without further purification. Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA) and used without further purification. Proton sponge (N,N,N′,N′-tetramethyl-1,8-naphthalenediamine) and all perfluorocarbon compounds were obtained from Aldrich (Milwaukee, WI). All experiments were performed using a prototype version of a Q TRAP mass spectrometer (Applied Biosystems/MDS Sciex, Concord, ON, Canada).29 The ion path is based on that of a triple quadrupole mass spectrometer with the last quadrupole rod array (Q3) configured to operate either as a conventional rf/dc mass filter or as an LIT with mass-selective axial ejection (MSAE).30 The Q TRAP was operated at a drive rf of 650 kHz with a nominal upper mass-to-charge limit of 4200 Th, when the standard MSAE frequency was used. All experiments were controlled by MS Expo 1.78 software provided by Applied Biosystems/MDS Sciex. To facilitate mutual trapping ion/ion reactions in Q2 of the instrument, the Q TRAP electronics were modified to allow superposition of an auxiliary rf signal to IQ3, which is the downstream containment lens of Q2 quadrupole array. This modification enabled simultaneous containment of both cations and anions in the z-dimension, while storage in the x- and y-dimensions was provided by the normal operation of the oscillating quadrupole field of the quadrupole array. The auxiliary rf signal is tunable over the ranges of 50-1000 kHz and 0-75 V0-p and was optimized for each ion/ion reaction experiment. In a typical ion/ion reaction experiment, SSI-generated ions of one polarity were injected axially into Q2, where either helium or nitrogen was present at a pressure of 6-8 mTorr. The ions were stored in Q2 for 50 ms, during which time the dc potentials on the lenses on either side of Q2, as well as on the Q2 rods themselves, were ramped to the same potential level while an auxiliary rf voltage was applied to IQ3. The dc potentials applied to the ion path prior to Q2 were then adjusted so that the SSI-generated ions of opposite polarity were introduced axially into Q2 at relatively low kinetic energies. During the subsequent mutual ion polarity storage period, the amplitude of the rf applied to the Q2 rods was adjusted to optimize the ion/ion reaction rate. In this instrument, the amplitude of the rf applied to Q2 is adjusted by varying the amplitude of the rf applied to Q3 because these two rod sets are capacitively coupled. After a defined period of (29) Hager, J. W. Rapid Commun. Mass Spectrom. 2002, 16, 512-526. (30) Londry, F. A.; Hager, J. W. J. Am. Soc. Mass Spectrom. 2003, 14, 11301147.

mutual storage, the unwanted polarity of ions was ejected from Q2 by applying attractive dc potentials to the Q2 containment lenses while the secondary rf signal applied to IQ3 was terminated. The product ions of interest were transferred from Q2 to Q3 and stored for 50 ms before they were subjected to MSAE using a supplementary rf signal at frequency 247 kHz or at a frequency selected to give the desired mass range extension. The spectra shown here were typically the average of 50-100 individual scans. RESULTS AND DISCUSSION Charge Reduction of Multiply Charged Positive Ions. The anions formed via glow discharge ionization of perfluorocarbons have been observed to react exclusively by proton transfer with a wide variety of peptide and protein cations without inducing fragmentation or giving rise to adduct species.11,13,14 Perfluorocarbons, however, are not readily ionized via SSI. Therefore, alternative reagents amenable to SSI are required for peptide/protein cation charge-state manipulation. Desirable characteristics of the reagent ions are as follows: (1) relatively high mass to allow for the mutual storage of the reagent anions as the mass-to-charge ratios of the cations increase due to charge state reduction, (2) high ion yields, (3) exclusive proton transfer with minimal tendency for adduct formation, and (4) minimal tendency to induce fragmentation. Based on these considerations, functionalized perfluorocarbn compounds were evaluated. 1H,1H,10H,10H-Perfluoro-1,10-decanediol (PFDD), perfluorotetradecanoic acid (PFTDA), perfluoro-tert-butyl alcohol (PFTB), nonadecafluoro-1-decanol (NDFD), and perfluoro-1-octanol (PFO) are five reagents for which results are described here. Each of the five compounds yielded strong singly charged anion signals under SSI conditions when present at concentrations of 10-50 µM in methanol. A variety of different ion types were noted, however, including deprotonated species, clusters, chloride adducts (in the case of PFO and NDFD) and fragments, the latter presumably having been formed in the atmospheric vacuum interface. In general, it is desirable that the reagent anions need not undergo a mass selection process. Therefore, in evaluating these potential reagents, the entire anion population formed via SSI was allowed to react with the polypeptide cations. Ion/ion reactivity of the five candidate reagents with the [M + 5H]5+ melittin cation are illustrated in Figure 1. All five perfluorocarbon compounds yielded ions that showed a high propensity for proton transfer. However, deprotonated PFTDA, and an ion at m/z 439 observed in the SSI mass spectrum of PFDD were observed to give rise to relatively small degrees of attachment. No such adducts were noted for anions derived from PFTB. The base peaks in the negative ion SSI spectra of NDFD and PFO correspond to [X + Cl-] ions, where X ) NDFD or PFO, and both of these species show small degrees of chloride transfer to the polypeptide cations. Factors that affect the extent to which adduct species are observed in ion/ion reactions in an electrodynamic ion trap have been discussed.31,32 In the case of the PFDD anions, the fact that the reagent is bifunctional allows for the possibility of more than one dipole interaction in the proton-transfer intermediate formed (31) He, M.; Emory, J.; McLuckey, S. A. Anal. Chem., in press, DOI: 10.1021/ ac0482312. (32) Wells, J. M.; Chrisman, P. A.; McLuckey, S. A. J. Am. Chem. Soc. 2003, 125, 7238-7249.

Figure 1. Post-ion/ion reaction mass spectra of [M + 5H]5+melittin with (a) PFDD, (b) PFTDA, (c) PFTB, (d) PFO, and (e) NDFD.

by the reactants. As the binding strength in the proton-transfer intermediate increases, the likelihood for the observation of adduct ions, formed via stabilization of the intermediate, also increases. In the case of PFTDA anions, while there is only one functional group, the strength of the dipole-dipole interaction between the carboxyl group and the charge site of the peptide is significantly greater than that for an hydroxyl group, as reflected in ab initio calculations with model systems.31 It is therefore not surprising that PFTDA anions shows a greater tendency for adduct formation than anions derived from PFTB, NDFD, or PFO, the functionalities of which are limited to a single hydroxyl group. In the cases of PFO and NDFD, for which [X + Cl-] species were formed via SSI, the small degree of chloride ion attachment is likely to arise from incomplete dissociation of the cation/anion complex. For those ion/ion encounters that proceed through a long-lived chemical intermediate, the following reaction sequence applies: -X

n+ - (n-1)+* MHn+ 98 n + [X + Cl ] f [MHn ‚XCl ] -HCl

(n-1)+ - (n-1)+* [MHn+ 98 MH(n-1) n Cl ]

Most ion/ion encounters proceed to the final proton-transfer product while some of the intermediate species that constitute chloride-transfer products survive. It is significant in Figure 1 that, while NDFD and PFO reduce +5 melittin into +1, no +1 was obvious in the case of PFTB, despite the fact that the +2 ion was highly abundant. This is likely due to the fact that electrodynamic ion traps have a limited massto-charge range over which ions can be stored simultaneously, and for a fixed electrode geometry and rf frequency, this is largely determined by the amplitude of the rf applied to the trap.33 In the case of PFTB, due to the comparatively low m/z value of the anions (m/z 235), the rf amplitude necessary for satisfactory storage of the anions is not effective in trapping +1 of melittin (m/z 2847). Higher rf amplitudes could be employed with anions derived from PFO (m/z 435) and from NDFD (m/z 535) such that storage of the +1 melittin ion was relatively efficient. The m/z storage range appears to be much wider for the 3-D (33) Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 1997, 69, 3760-3766.

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Figure 2. Mass spectra derived from sonic spray of methanol solution containing 10 µM ubiquitin and 50 µM PFO with 0.5% acetic acid added: (a) in positive detection mode and (b) in negative detection mode. (c) Post-ion/ion and (d) ion parking (31.1 kHz, 2.0 V0-p) mass spectra were acquired after 200-ms reaction of ions shown in (a) and (b).

electrodynamic ion traps in our laboratory than for the Q2 LIT used in these studies. This may be due to the use of rf on only one side of the LIT. The issue of m/z storage range in both linear and 3-D ion traps is currently under investigation. In any case, NDFD and PFO were chosen for further study because of their relatively high m/z anions and their strong tendency for proton transfer. Figure 2 shows results of ion/ion reaction experiments involving bovine ubiquitin cations and PFO anions, both formed via SSI of a methanol solution (0.5% acetic acid) containing 10 µM bovine ubiquitin and 50 µM PFO. Panels a and b in Figure 2 show the pre-ion/ion reaction mass spectra generated in each polarity mode and reflect the identities of the ions generated simultaneously by sonic spray. The post-ion/ion mass spectrum (Figure 2c) was acquired at an axial ejection frequency of 83 kHz after the entire ubiquitin charge-state distribution was subjected to ion/ion reaction with PFO anions for 200 ms. The +1 charge state of ubiquitin was not observed, which, in this case, was subsequently found to be due to a problem in the rf supply that limited its amplitude to a value too low to allow the +1 ion to undergo MSAE. The concentration of ions dispersed over multiple charge states into a single charge state is a useful capability afforded by conducting ion/ion reactions in an electrodynamic ion trap. This technique, referred to as ion parking,34 is based upon the inhibition of the reaction rate of the charge state of interest, while the reaction rates of other charge states are largely unaffected. This can be done by applying to the ion trap electrodes a supplementary rf voltage that is in resonance with the ion of interest. Under the current instrument setup, a dipolar auxiliary signal is coupled directly to one set of opposing poles of Q3 quadrupole array. Due to the capacitive coupling of Q3 and Q2, the rf signal is also applied across opposing rods of Q2, although at a lower amplitude. Ion parking experiments were performed on the +3, +4, and +5 (34) McLuckey, S. A.; Reid, G. E.; Wells, J. M. Anal. Chem. 2002, 74, 336346.

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Figure 3. Mass spectra derived from sonic spray of water solution containing 20 µM 5′-d(T)6-3′ and 10 µM proton sponge: (a) isolated -4 charge state of 5′-d(T)6-3′ with storage in Q2 for 400 ms in negative detection mode and (b) proton sponge in positive detection mode. (c) Post-ion/ion spectrum was acquired after 400 ms reaction of ions shown in (a) and (b).

charge states of ubiquitin. In all cases, significant concentration of signal in the ion for which the resonance excitation of signal was most closely tuned was observed. Figure 2d shows results of the ion parking experiment applied to the +3 charge-state ions using the same ion/ion reaction conditions as in Figure 2c except that a resonance excitation signal at 31.1 kHz, 2.0 V0-p was applied for 200 ms to an opposing set of rods in the Q2 LIT. Charge Reduction of Multiply Charged Anions. Ion/ion reactions of multiply charged oligonucleotide anions have been investigated using several different combinations of ion sources with reagent cations such as the oxygen cation, ionized xenon, protonated isobutylene, and protonated benzoquinoline.15,16,35 While oxygen cations react with oligonucleotide anions via electron transfer, and have been observed to lead to fragmentation, much less fragmentation has been observed with protonated isobutylene and protonated benzoquinoline as proton-transfer reagents. In this work, the proton sponge (N,N,N′,N′-tetramethyl1,8-naphthalenediamine) was evaluated as a proton-transfer reagent. Mass spectra derived from sonic spray of a water solution containing 20 µM 5′-d(T)6-3′ and 10 µM proton sponge are shown in negative mode after isolation of the -4 charge state of 5′-d(T)6-3′ and tapping in Q2 LIT for 400 ms (Figure 3a), and in positive detection mode without use of ion isolation (Figure 3b). The post-ion/ion reaction spectrum (Figure 3c) was acquired after a mutual ion storage period of 400 ms in Q2 in the presence of nitrogen (6 mTorr). A secondary rf (120 kHz, 70 V0-p) voltage was applied to IQ3 only during the ion/ion reaction period. In Figure 3c, [M - 4H]4- anions were predominantly reduced to -1 charge state with no apparent adduct formation or fragmentation. Ion/ion proton-transfer reactions can be helpful in resolving ambiguities in the charge states of product ions derived from fragmentation of multiply charged parent ions,36-39 particularly (35) Herron, W. J.; Goeringer, D. E.; Mcluckey, S. A. J. Am. Soc. Mass Spectrom. 1995, 6, 529-532. (36) Reid, G. E.; Wu, J.; Chrisman, P. A.; Wells, J. M.; McLuckey, S. A. Anal. Chem. 2001, 73, 3274-3281. (37) Reid, G. E.; McLuckey, S. A. J. Mass Spectrom. 2002, 37, 663-675. (38) Hogan, J. M.; McLuckey, S. A. J. Mass Spectrom. 2003, 38, 245-256. (39) Amunugama, R.; Hogan, J. M.; Newton, K. A.; McLuckey, S. A. Anal. Chem. 2004, 76, 720-727.

Figure 4. Pre- (a) and post-ion/ion (b) MS/MS mass spectra of [M - 4H]4- 5′-d(T)6-3′ via beam-type CID at collision energy of 164 eV. The post-ion/ion MS/MS spectrum was acquired after a 300-ms ion/ ion reaction period.

when the resolving power of the mass analyzer is too low to separate the isotope peaks of the products. A straightforward approach to this problem is to subject all product ions, as well as residual parent ions, to a single ion/ion reaction period that converts most of the products to singly charged ions. This approach is effective in resolving all charge-state ambiguities with a single ion/ion reaction and also avoids charge-state-dependent detection efficiency issues in assessing the relative contributions of various dissociation channels. An example is given here with both the analyte ions and the charge manipulation reagents formed simultaneously via SSI. The Q TRAP instrument allows for both beam-type and in-trap collisional activation. The [M 4H]4- parent ions derived from 5′-d(T)6-3′ were subjected to both forms of collisional activation. For the beam-type experiment, the parent ions were selected by Q1 in the mass-resolving mode, and then subjected to beam-type collision-induced dissociation (CID) in the high-pressure Q2 collision cell (laboratory collision energy: 164 eV, collision gas: nitrogen, pressure: ∼8 mTorr). The product ion spectrum formed in this way is shown in Figure 4a. After the CID fragments were cooled in Q2 for 50 ms, proton sponge cations were injected axially into Q2, while a secondary rf on IQ3 was turned on for the succeeding 300 ms ion/ion reaction period. When most of the multiply charge ions were reduced to the -1 charge state, the secondary rf signal on IQ3 was terminated and the post-ion/ion reaction products were transferred from Q2 to Q3 LIT to be mass analyzed via MSAE. The resulting spectrum is given in Figure 4b. In-trap collisional activation in Q2 was effected by applying a supplementary rf voltage (74.7 kHz, 1.7 V0-p) across one set of opposing rods in resonance with the parent ion, which was selected by Q1 and injected into Q2 at relatively low energy to avoid beam-type collisional activation. The pre-ion/ion product ion spectrum obtained in this way is shown in Figure 5a. The postion/ion reaction product ion spectrum in Figure 5b was obtained using an ion/ion reaction procedure very similar to that described for the beam-type collisional activation experiment leading to Figure 4b. From the data in Figures 4 and 5, it is clear that, for both beamtype and in-trap CID, the major fragmentation channels are base

Figure 5. Pre- (a) and post-ion/ion (b) MS/MS mass spectra of [M - 4H]4- 5′-d(T)6-3′ via in-trap CID (dipolar excitation: 74.3 kHz, 1.3 V0-p, 200 ms). The post-ion/ion MS/MS spectrum was acquired after a 300-ms ion/ion reaction period.

Figure 6. Mass spectra derived from sonic spray of water containing 10 µM insulin and 30 µM DDDD: (a) isolated +5 charge state of insulin with 150-ms storage in Q2 in positive detection mode and (b) DDDD in negative mode. (c) Post-ion/ion mass spectra were acquired after 150-ms reaction of ions shown in (a) and (b).

loss, e.g., either charged or uncharged, and the subsequent fragmentation of the 3′ C-O bond of the sugar from which the base was lost, which gives complementary w-type and (a-B)-type ions. Although both activation methods provide extensive sequence information, more product ions are observed in the beamtype CID spectra (Figure 4) than from the in-trap CID spectra (Figure 5). This is due in part to the fact that a lower Q2 rf amplitude could be used to collect the beam-type CID spectra, thereby allowing for the observation of the w1- and (a2-T)- ions. Complex Formation. It has recently been noted that ion/ion reactions can serve as a means for the synthesis of bio-ion complexes in the gas phase.32,40 Analytical applications of this capability have not yet been developed, but it has already proved to be useful in the characterization of gas-phase complexes formed initially in solution by comparing the dissociation behaviors of the ions formed in gas phase with those formed initially in solution and subsequently transferred to the gas phase. It is of interest, (40) Gunawardena, H. P.; McLuckey, S. A. J. Mass Spectrom. 2004, 39, 630638.

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Figure 7. (a) Abundance of melittin cations and NDFD anions as a function of NDFD concentration from sonic spray of the solutions, where melittin was kept at 5 µM while the concentration of NDFD was increased. (b) Abundance of ubiquitin cations and NDFD anions as a function of NDFD concentration from sonic spray of the solutions, where ubiquitin was kept at 10 µM while the concentration of NDFD was increased.

therefore, to examine the potential for SSI as a source of both ionic reactants for the synthesis of a complex in the gas phase. Given the limited m/z range of the current approach for mutual ion storage in Q2, a relatively small system was chosen to evaluate the possibility for gas-phase complex synthesis. Bovine insulin, denoted as I, was mixed with the tetrapeptide DDDD, denoted as D, to give concentrations of 10 and 30 µM, respectively, in deionized water and subjected to sonic spray ionization. The [M + 5H]5+ ions of insulin, I5+, was isolated and reacted with the subsequently injected [M - H]- anions of DDDD, denoted as D-, in the Q2 LIT (bath gas, nitrogen at 7 mTorr; reaction time, 150 ms; secondary rf applied on IQ3 lens, 90 kHz; 70 V0-p). Figure 6 shows the relevant pre- and post-ion/ion reaction mass spectra. The predominant peak in the post-ion/ion spectrum is the complex (D + I)4+, which is formed directly by the condensation of the two reactants. The D+, I3+, and I4+ products can also be formed via a single ion/ion encounter. All three ions, for example, can arise from the breakup of the (D + I)4+ ion. All other products likely require the involvement of at least two ion/ion reaction steps. The (3D + I)3+ is particularly curious in that it is not clear how it can be formed from the two nominal reactants. It might be that the ion population labeled as D- also contained some multiply charged multimers at the same nominal mass-to-charge ratio. If so, several two step ion/ion reaction pathways can be envisioned to give rise to this product. In any case, this experiment demonstrates the formation of a peptide/peptide complex in the gas phase from two peptide components present in the same solution and undergoing ionization simultaneously. Matrix Effects. For all the ion/ion reactions described herein, two species were mixed in the solution to generate ions of each polarity by the single sonic spray source. It is therefore of interest to examine how the presence of reagent species in solution affects the ionization yield of the analyte species. In the case of charge manipulation of multiply charged cations, a perfluorocarbon compound was mixed with the protein/peptide of interest. To examine the possible effect on the addition of a perfluorocarbon to the ion yield of a peptide, melittin and NDFD were chosen as the test pair. A series of experiments were conducted in which melittin was maintained at a concentration of 5 µM in methanol, while NDFD was added to each solution to yield concentrations over a range of 5-200 µM. Both the total abundances of the NDFD anions and the total abundances of the melittin cations are plotted as a function of the concentration of NDFD in Figure 7a. No obvious signal suppression was noted for melittin through 3688

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the tested range of NDFD concentrations. Similar experiments were conducted for 10 µM ubiquitin and NDFD, as summarized in Figure 7b, with similar findings. For PFO, analogous experiments were conducted with similar results. Experiments were also performed on oligonucleotides with a range of proton sponge concentrations. No obvious evidence for signal suppression of the oligonucleotide anions was found when the proton sponge concentration was increased to as high as 200 µM (data not shown). Based on the apparent signal saturation noted for reagents ions at the higher concentrations, it is clear that there is no advantage to using such high concentrations. In any case, for the species described in this report, there appears to be little signal suppression of the analyte ions encountered by the addition of the reagent species intended to give rise to oppositely charged ions. CONCLUSIONS Sonic spray can be used for the simultaneous formation of both cationic and anionic reactants for subsequent ion/ion reaction experiments. Several reagents, including PFO and NDFD, have been identified that are effective in removing protons from multiply protonated peptides and proteins. The proton sponge was found to be effective in protonating multiply deprotonated oligonucleotides. The presence of these species in the sonic spray solutions, over the concentration ranges investigated, appears to have no measurable impact on the ion yields for polypeptide or oligonucleotide analyte species. The formation of a peptide/peptide complex via the ion/ion reaction of multiply protonated insulin with a singly deprotonated tetrapeptide was also demonstrated. All experiments were conducted within a linear ion trap of a hybrid triple quadrupole/linear ion trap mass spectrometer with axial trapping produced by applying auxiliary rf to one end lens of the quadrupole array. Important applications of ion/ion reactions involving reagents formed simultaneously from a single source were demonstrated, such as parent ion charge reduction, ion parking, simplification of MS/MS spectra, and complex formation. The key instrumental requirement for these types of studies is that the appropriate voltage settings for transmission of the oppositely charged ions can be applied in the proper sequence to allow for the accumulation of each reactant ion polarity. Since SSI can be readily coupled to essentially any spray ionization interface and, in general, can be expected to be able to generate the kinds of ions normally formed via electrospray, it can, in principle, be used for virtually any of the proton-transfer ion/ion reaction experi-

ments that have thus far been demonstrated by the use of two distinct ionization sources. ACKNOWLEDGMENT This work was sponsored by MDS Sciex, an Industrial Associate of the Department of Chemistry. We acknowledge Dr. James W. Hager and Dr. Frank A. Londry of MDS Sciex for helpful discussions and Adam Lau of MDS Sciex for providing custom instrument control software. We also thank Dr. Robert E. Santini

of the Jonathan Amy Facility for Chemical Instrumentation for helpful advice. Dr. Zoltan Takats and Prof. Graham Cooks are acknowledged for guidance on the construction and operation of the SSI source. Y.X. thanks Dr. Jin Wu and Paul A. Chrisman for their initial contributions to this work. Received for review December 9, 2004. Accepted March 22, 2005. AC0481811

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