Charge-Remote Fragmentation of Odd-Electron Peptide Ions

Aug 4, 2007 - We have found that charge-remote processes are responsible for a variety of side-chain losses from the precursor ion and some backbone ...
0 downloads 0 Views 179KB Size
Download PDF
Anal. Chem. 2007, 79, 6607-6614

Charge-Remote Fragmentation of Odd-Electron Peptide Ions Julia Laskin,*,† Zhibo Yang,† Corey Lam,‡ and Ivan K. Chu*,‡

Fundamental Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, and Department of Chemistry, University of Hong Kong, Hong Kong, China

Comparison between the gas-phase fragmentation of oddelectron M+•, [M + H]2+•, and [M - 2H]-• ions of model peptides suggests that charge-remote radical-driven fragmentation pathways play an important role in the dissociation of odd-electron peptide ions. We have found that charge-remote processes are responsible for a variety of side-chain losses from the precursor ion and some backbone fragmentation. These fragmentation pathways most likely involve hydrogen abstraction by the radical site that initiates subsequent cleavages. These findings are generally relevant to our understanding of the fragmentation patterns of odd-electron peptide ions produced through various approaches including the capture of lowenergy electrons, electron detachment, and electron transfer. Structural characterization of molecules using tandem mass spectrometry (MS/MS) relies on the structure-specific fragmentation of mass-selected ions in a mass spectrometer.1 Decomposition of gas-phase ions is typically directed by the charge site that efficiently catalyzes cleavage of an adjacent bond. Alternatively, bond cleavage may occur at a site that is physically remote from the charge site, resulting in charge-remote fragmentation.2-4 During the past decade, MS/MS became an invaluable tool for identification of large biomolecules. Peptides and proteins are introduced into the gas phase using soft ionization techniquess such as electrospray ionization,5 matrix-assisted laser desorption ionization,6 or other soft ionization techniques7 that usually result in very little fragmentation of the precursor ion. These ions can be further excited using a variety of ion activation techniques8,9 * Corresponding authors. E-mail: Julia. [email protected]; Pacific Northwest National Laboratory, P.O. Box 999 K8-88, Richland, WA 99352. E-mail: ivankchu@ hku.hk; Department of Chemistry, the University of Hong Kong, Pokfulam, Hong Kong. † Pacific Northwest National Laboratory. ‡ University of Hong Kong. (1) Shukla, A. K.; Futrell, J. H. J. Mass Spectrom. 2000, 35, 1069-1090. (2) Cheng, C. F.; Gross, M. L. Mass Spectrom. Rev. 2000, 19, 398-420. (3) Gross, M. L. Int. J. Mass Spectrom. 2000, 200, 611-624. (4) Claeys, M.; Nizigiyimana, L.; Van Den Heuvel, H.; Vedernikova, I.; Haemers, A. J. Mass Spectrom. 1998, 33, 631-643. (5) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (6) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, A1193-A1202. (7) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311, 1566-1570. (8) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. Rev. 2005, 24, 135167. (9) Laskin, J.; Futrell, J. H. Mass Spectrom. Rev. 2003, 22, 158-181. 10.1021/ac070777b CCC: $37.00 Published on Web 08/04/2007

© 2007 American Chemical Society

to produce a sufficient amount of fragment species on the time scale of a mass spectrometer for successful identification. Low-energy dissociation of protonated peptide ions is dominated by charge-directed processes. Mechanisms of peptide fragmentation are commonly discussed within the framework of the “mobile proton” model.10,11 It is assumed that dissociation is initiated by the transfer of the ionizing proton to the amide nitrogen or the carbonyl oxygen atom of the corresponding peptide bond. The gas-phase fragmentation behavior of peptide ions in mass spectrometry has been summarized in a number of commentary articles12-15 and recent reviews.16,17 In general, charge-remote fragmentation of even-electron peptide ions occurs at higher internal excitations that can be achieved using highenergy collisions with neutral molecules,18,19 absorption of VUV light,20 or ion-surface collisions.21 However, it has been suggested that selective fragmentation at acidic residues observed in lowenergy, collision-induced dissociation (CID) spectra of protonated peptides occurs through a charge-remote process in which the ionizing proton resides on the highly basic arginine side chain and does not play an active role in the cleavage.22 Gas-phase dissociation of odd-electron peptide ions has attracted considerable attention because of the unique fragmentation behavior of hydrogen-rich radical cations, [M + nH](n-1)+•, produced through capture of low-energy electrons by multiply protonated precursors.23 While the fragmentation of even-electron (10) Burlet, O.; Orkiszewski, R. S.; Ballard, K. D.; Gaskell, S. J. Rapid Commun. Mass Spectrom. 1992, 6, 658-662. (11) Dongre, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, V. H. J. Am. Chem. Soc. 1996, 118, 8365-8374. (12) O’Hair, R. A. J. J. Mass Spectrom. 2000, 35, 1377-1381. (13) Polce, M. J.; Ren, D.; Wesdemiotis, C. J. Mass Spectrom. 2000, 35, 13911398. (14) Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399-1406. (15) Schlosser, A.; Lehmann, W. D. J. Mass Spectrom. 2000, 35, 1382-1390. (16) Paizs, B.; Suhai, S. Mass Spectrom. Rev. 2005, 24, 508-548. (17) Wysocki, V. H.; Cheng, G.; Zhang, Q.; Herrmann, K. A.; Beardsley, R. L.; Hilderbrand, A. E. Peptide Fragmentation Overview. In Principles of Mass Spectrometry Applied to Biomolecules; Laskin, J., Lifshitz, C., Eds.; John Wiley and Sons: New York, 2006. (18) Johnson, R. S.; Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Processes 1988, 86, 137-154. (19) Downard, K. M.; Biemann, K. J. Am. Soc. Mass Spectrom. 1994, 5, 966975. (20) Cui, W. D.; Thompson, M. S.; Reilly, J. P. J. Am. Soc. Mass Spectrom. 2005, 16, 1384-1398. (21) McCormack, A. L.; Somogyi, A.; Dongre, A. R.; Wysocki, V. H. Anal. Chem. 1993, 65, 2859-2872. (22) Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. J. Am. Chem. Soc. 1999, 121, 51425154.

Analytical Chemistry, Vol. 79, No. 17, September 1, 2007 6607

peptide ions is dominated by the cleavage of amide bonds, electron capture dissociation (ECD) and the related technique of electrontransfer dissociation (ETD) are characterized by cleavages of N-CR bonds.24-28 Other types of odd-electron peptide ions include peptide radical cations, M+•, produced by CID of complexes of transition metals with peptides 29-32 or through free-radical-initiated reactions,33,34 and hydrogen-deficient radical anions, [M - nH](n-1)-•, produced through electron detachment from multiply deprotonated ions (EDD).35,36 It has been suggested that distonic ions, in which the charge site is separated from the radical site, play an important role in the dissociation of M+• ions.29,37 DFT calculations performed by Siu and co-workers suggested that the lowest energy structure of the radical cation of triglycine is a distonic ion, in which the positive charge is localized on the R-carbonyl oxygen atom of the residue, to which the proton is attached, while the unpaired electron is localized on the R-carbon of the first residue.38 O’Hair and co-workers have described in detail the radical-initiated reactions that occur in the dissociation of a variety of small glycine-containing peptides.39 They suggested that fragmentation of peptide radical cations is determined by the competition between charge-directed processes involving mobile proton and radical-driven processes. They further demonstrated that the fragmentation can be restricted to radical-driven processes alone for peptides incorporating a fixed-charge group.40 (23) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (24) McLafferty, F. W.; Horn, D. M.; Breuker, K.; Ge, Y.; Lewis, M. A.; Cerda, B.; Zubarev, R. A.; Carpenter, B. K. J. Am. Soc. Mass Spectrom. 2001, 12, 245-249. (25) Zubarev, R. A. Mass Spectrom. Rev. 2003, 22, 57-77. (26) Zubarev, R. A. Electron Capture Dissociation and Other Ion-Electron Fragmentation Reactions. In Principles of Mass Spectrometry Applied to Biomolecules; Laskin, J., Lifshitz, C., Eds.; John Wiley and Sons: New York, 2006. (27) 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. (28) Coon, J. J.; Shabanowitz, J.; Hunt, D. F.; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2005, 16, 880-882. (29) (a) Chu, I. K.; Rodriquez, C. F.; Lau, T. C.; Hopkinson, A. C.; Siu, K. W. M. J. Phys. Chem. B 2000, 104, 3393-3397. (b) Chu, I. K.; Rodriquez, C. F.; Lau, T. C.; Hopkinson, A. C.; Siu, K. W. M. J. Am. Soc. Mass Spectrom. 2001, 12, 1114-1119. (c) Ke, Y.; Verkerk, U. H.; Shek, P. Y. I.; Hopkinson, A. C.; Siu, K. W. M. J. Phys. Chem. B 2006, 110, 8517-8523. (d) Chu, I. K.; Siu, S. O.; Lam, C. N. W.; Chan, J. C. Y.; Rodriquez, C. F. Rapid Commun. Mass Spectrom. 2004, 18, 1798-1802. (e) Lam, C. N. W.; Siu, S. O.; Orlova, G.; Chu, I. K. Rapid Commun. Mass Spectrom. 2005, 20, 790-796. (f) Lam, C. N. W.; Ruan, E. D. L.; Ma, C. Y.; Chu, I. K. J. Mass Spectrom. 2006, 41, 931-938. (30) Chu, I. K.; Lam, C. N. W.; Siu, S. O. J. Am. Soc. Mass Spectrom. 2005, 16, 763-771. (31) Barlow, C. K.; McFadyen, W. D.; O’Hair, R. A. J. J. Am. Chem. Soc. 2005, 127, 6109-6115. (32) Wee, S.; Mortimer, A.; Moran, D.; Wright, A.; Barlow, C. K.; O’Hair, R. A. J.; Radom, L.; Easton, C. J. Chem. Commun. 2006, 4233-4235. (33) Masterson, D. S.; Yin, H. Y.; Chacon, A.; Hachey, D. L.; Norris, J. L.; Porter, N. A. J. Am. Chem. Soc. 2004, 126, 720-721. (34) Hodyss, R.; Cox, H. A.; Beauchamp, J. L. J. Am. Chem. Soc. 2005, 127, 12436-12437. (35) Kjeldsen, F.; Silivra, O. A.; Ivonin, I. A.; Haselmann, K. F.; Gorshkov, M.; Zubarev, R. A. Chem. Eur. J. 2005, 11, 1803-1812. (36) Lam, C. N. W.; Chu, I. K. J. Am. Soc. Mass Spectrom. 2006, 17, 12491257. (37) Hopkinson, A. C.; Siu, K. W. M. Peptide Radical Cations. In Principles of Mass Spectrometry Applied to Biomolecules; Laskin, J., Lifshitz, C., Eds.; John Wiley and Sons: New York, 2006. (38) Bagheri-Majdi, E.; Ke, Y. Y.; Orlova, G.; Chu, I. K.; Hopkinson, A. C.; Siu, K. W. M. J. Phys. Chem. B 2004, 108, 11170-11181. (39) Wee, S.; O’Hair, R. A. J.; McFadyen, W. D. Int. J. Mass Spectrom. 2006, 249, 171-183.

6608

Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

In this paper, we present the experimental evidence for chargeremote radical-initiated fragmentation pathways of odd-electron peptide ions through a comparison of the gas-phase fragmentation of M+•, [M + H]2+•, and [M - 2H]-• ions produced by redox chemistry of CoIII(salen)+ and CuII(terpy)2+ complexes incorporating neutral, singly protonated, or doubly deprotonated peptides. The characteristic charge-remote fragmentation behavior observed for these species is generally relevant to our understanding of the gas-phase fragmentation of a variety of odd-electron peptide ions produced using other techniques (e.g., ECD, ETD, or EDD). EXPERIMENTAL SECTION FT-ICR Experiments. Experiments were conducted using a specially fabricated 6-T FT-ICR mass spectrometer, which is described elsewhere.41 The instrument is equipped with a hightransmission electrospray source, consisting of an ion funnel interface42 followed by three quadrupoles that provide a pressure drop and ion bunching, mass selection, and ion storage, respectively. The SID target was introduced through a vacuum interlock assembly and was positioned at the rear trapping plate of the ICR cell. Ions were electrosprayed, at atmospheric pressure, into the end of a heated stainless steel capillary tube. The ion funnel that follows the capillary provided highly efficient ion transfer into the high-vacuum region of the mass spectrometer. Three quadrupoles following the ion funnel provided collisional focusing, mass selection of the ion of interest, and accumulation of ions external to the ICR cell. The third (accumulation) quadrupole was held at an elevated pressure (∼2 × 10-3 Torr) for collisional relaxation of any internal energy possessed by ions generated through electrospray ionization prior to their injection into the ICR cell. Radical cations, M+•, were produced through in-source fragmentation of the corresponding positively charged [CoIII(salen)M]+ complexes, while radical anions, [M - 2H]-•, were produced from negatively charged [CoIII(salen)(M - 2H)]precursors. Doubly charged radical cations, [M + H]2+•, were produced from triply charged [CuII(terpy)(M + H)]3+• complexes. Mass-selected, odd-electron ions were accumulated for 1-5 s, extracted from the third quadrupole, and transferred into the ICR cell. For SID experiments, ions were allowed to collide with the surface. Scattered ions were captured by raising the potentials on the front and rear trapping plates of the ICR cell by 10-20 V. Time-resolved mass spectra were acquired by varying the delay between the gated trapping and the excitation/detection event (the reaction delay) from 1 ms to 1 s. Immediately following the fragmentation delay, ions were excited through a broadband chirp and detected. The collision energy is defined by the difference between the potential applied to the accumulation quadrupole and the potential applied to the rear trapping plate and the SID target. The ICR cell can be offset above or below ground by as much as (150 V. Lowering the ICR cell below ground while keeping the potential on the third quadrupole fixed increases collision energy for positive ions. (40) Karnezis, A.; Barlow, C. K.; O’Hair, R. A. J.; McFadyen, W. D. Rapid Commun. Mass Spectrom. 2006, 20, 2865-2870. (41) Laskin, J.; Denisov, E. V.; Shukla, A. K.; Barlow, S. E.; Futrell, J. H. Anal. Chem. 2002, 74, 3255-3261. (42) Shaffer, S. A.; Tang, K. Q.; Anderson, G. A.; Prior, D. C.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1997, 11, 1813-1817.

For CID experiments, ion were transferred into the ICR cell and captured using gated trapping in a 6-8 V potential well. All unwanted ions were ejected by applying a stored waveform inverse Fourier transform excitation. Sustained off-resonance irradiation (SORI)-CID experiments43 were performed on the isolated single isotope peak of the ion of interest. Collision gas (Ar) was introduced into the cell using a pulsed valve. The isolated precursor ions were radially excited slightly off resonance for 40 ms with maximum center-of-mass collision energies in the range of 0.1-0.9 eV by changing the peak-to-peak voltage applied to the excitation plates. After a 10-s pumping delay, the ions in the cell were excited for detection by a broadband chirp excitation. The maximum kinetic energy (peak-peak) achieved in SORI-CID was calculated as reported previously.44 Experimental control was accomplished using the MIDAS data station developed by Marshall and co-workers at the National High Magnetic Field Laboratory.45 MIDAS is used to control the voltages and timing of the source and transfer optics, as well as ion manipulation in the ICR cell. Reactions delays of 1, 5, 10, and 50 ms and 0.1 and 1 s were studied. Typical experiments involved changing the collision energy across a relatively wide ranges (11.5-79.5 and 15.5-85.5 eV for the M+• and [M - 2H]-• ions of RVYIHPF, respectively; 15.5-71.2 and 25.5-106.5 eV for the M+• and [M - 2H]-• ions of HVYIHPF, respectively) in increments of 2-3 eV at each of the six fragmentation delays. Time-dependent fragmentation efficiency curves were constructed from experimental mass spectra by plotting the relative abundance of a selected fragment as a function of collision energy for each delay time. SID Target. A self-assembled monolayer surface of 1-dodecanethiol (HSAM) was prepared on a single gold {111} crystal (Monocrystals, Richmond Heights, OH) using a standard procedure. The target was cleaned in a UV cleaner (model 135500, Boekel Industries Inc., Feasterville, PA) for 10 min and left to stand in a solution of 1-dodecanethiol for 10 h. The target was removed from the SAM solution and washed ultrasonically in ethanol for 10 min to remove extra layers. Ion Trap Experiments. Ion trap experiments were conducted using a quadrupole ion trap mass spectrometer (Finnigan LCQ, ThermoFinnigan, San Jose, CA). Samples were continuously infused at a rate of 5 µL/min into the pneumatically assisted electrospray probe using air as the nebulizer gas. CID spectra were acquired using helium as the collision gas. The injection and activation times for CID in the ion trap were 200 and 30 ms, respectively; the amplitude of the excitation was optimized for each experiment. Chemicals. All chemicals and reagents used were commercially available (Sigma-Aldrich, St. Louis, MO; Bachem, King of Prussia, PA). Angiotensin III (RVYIHPF) and its derivative (HVYIHPF) were synthesized according to literature procedures.46 Fmoc-protected amino acids and the Wang resin were purchased from Advanced ChemTech (Louisville, KY). Tyr5-bradykinin and Tyr8-bradykinin were purchased from the American Peptide Company (Sunnyvale, CA) and used as received. N,N′-Ethylenebis(43) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (44) Laskin, J.; Byrd, M.; Futrell, J. Int. J. Mass Spectrom. 2000, 196, 285-302. (45) Senko, M. W.; Canterbury, J. D.; Guan, S. H.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1839-1844. (46) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical Approach; Oxford: New York, 2000.

Figure 1. SORI-CID spectra of the (a) M+• ion and (b) [M - 2H]-• ion of RVYIHPF; (c) fragment distribution from the 75-eV SID spectrum of the [M - 2H]-• ion of RVYIHPF. Common noise peaks are denoted with an asterisk.

(salicylideneaminato) was prepared according to the literature procedure.47,48 The [Cu(II)(terpy)]ClO4 complex (terpy ) 2,2′:6′,2′′-terpyridine) was synthesized according to the standard experimental procedure.49 [Cobalt(III)(salen)]Cl complex [salen ) N,N′-ethylenebis(salicylideneaminato)] was synthesized using methods similar to a published method,50 but with cobalt(II) chloride replacing manganese(II) acetate as the metal salt. Samples typically comprised a 600 µM metal-salen complex and 50 µM peptide in a water/methanol (50:50) solution. A syringe pump (Cole Parmer, Vernon Hills, IL) was used for direct infusion of the electrospray samples at a flow rate of 30 µL/h. RESULTS AND DISCUSSION Detailed description of the fragmentation behavior of positive and negative radical cations is beyond the scope of this paper and will be presented in our forthcoming publications. Here we will compare the fragmentation patterns of M+• and [M - 2H]-• ions obtained for a number of model peptides and examine the role of radical-driven charge-remote processes in the dissociation of these species. Figure 1 shows a comparison of the SORI-CID spectra obtained for the M+• and [M - 2H]-• ions of angiotensin III (RVYIHPF). The spectrum obtained in the positive mode is dominated by losses of small molecules from peptide side chains and the formation of an+ ions through CR-C bond cleavage. The most abundant neutral losses correspond to the losses of CO2 from the C-terminal carboxyl group and loss of p-quinomethide (C7H6O, 106.0418) from the tyrosine side chain. Other fragments (47) Archer, R. D.; Chen, H. Inorg. Chem. 1998, 37, 2089-2095. (48) Chen, H.; Cronin, J. A.; Archer, R. D. Macromolecules 1994, 27, 21742180. (49) Henke, W.; Kremer, S.; Reinen, D. Inorg. Chem. 1983, 22, 2858-2863. (50) Varkey, S. P.; Ratnasamy, C.; Ratnasamy, P. J. Mol. Catal. A 1998, 135, 295-306.

Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

6609

6610

Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

[a5-106] + z4+• [b5-H] +• y2+

Major fragments are presented as boldface text. b Molecular weight of the neutral fragment. c Residue from which the corresponding side chain loss occurred.

[a5-106] + z4+• [b5-H] +• y2+ [a5-106] + z4+• [b5-H] +• y2+

M+• a5+ M+• a5+

[M-2H]-• [x2-H]-• [a5-2H][a5-2H-106][z4-2H][b5-3H]-•

C7H6O (106.0418), Y C3H8N3• (86.0718), R C2H5• (29.0391), I H2O NH3 C7H6Oa (106.0418)b, Yc C3H8N3• (86.0718), R C2H5• (29.0391), I H2O NH3

a

[a5-106] + z4+• [b5-H] +• y 2+

[M-2H]-• [x2-H]-• [a5-2H][a5-2H-106][z4-2H][b5-3H]-• M+• a 5+ M+• a5+

Common Backbone Fragments M+• [M-2H]-• [M+H]2+• +• z3 [z3-2H]-• z3+• z4+• [z4-2H]-• z4+• z5+• [z5-4H]-• z5+• z6+• [z6-2H]-• z6+• [x5+H]+• [x5-H]-• [x5+H]+• [M-2H]-• [x2-H]-• [a5-2H][a5-2H-106][z4-2H][b5-3H]-•

Common Neutral Losses C7H6O (106), Y C3H8N3• (86), R or [C3H8N3•+H]+ (87) C2H5• (29), I

C7H6O (106.0418), Y C4H5N2• (81.0453), H C2H5• (29.0391), I H2O NH3

[M-2H]-• [x2-H]-• [a5-2H][a5-2H-106][z4-2H][b5-3H]-•

C7H6O (106.0418), Y C4H5N2• (81.0453), H C2H5• (29.0391), I H2O NH3

SID HVYIHPF SORI-CID ion trap RVYIHPF SID SORI-CID

(51) Wee, S.; O’Hair, R. A. J.; McFadyen, W. D. Int. J. Mass Spectrom. 2004, 234, 101-122; (52) Chu, I. K.; Lam, C. N. W. J. Am. Soc. Mass Spectrom. 2005, 16, 17951804. (53) Biemann, K. Methods Enzymol. 1990, 193, 886-887. (54) Bowie, J. H.; Brinkworth, C. S.; Dua, S. Mass Spectrom. Rev. 2002, 21, 87107. (55) Harrison, A. G. J. Am. Soc. Mass Spectrom. 2001, 12, 1-13. (56) Clipston, N. L.; Jai-nhuknan, J.; Cassady, C. J. Int. J. Mass Spectrom. 2003, 222, 363-381. (57) Laskin, J.; Denisov, E.; Futrell, J. J. Phys. Chem. B 2001, 105, 1895-1900. (58) Laskin, J.; Futrell, J. H. Mass Spectrom. Rev. 2003, 22, 158-181.

Table 1. Common Neutral Losses and Backbone Fragments Observed in SORI-CID and SID Experiments for the M+• and [M - 2H]-• Ions of RVYIHPF and HVYIHPFa

include the loss of NH3, the loss of C2H5• (29.0391) from isoleucine, the loss of C4H9N3 (99.0796) and C3H8N3• (86.0718) from arginine, the loss of COOH• from the C-terminus, and a few consecutive small molecule losses. These results are generally consistent with the fragmentation behavior of other peptide radical cations.,37,39,51,52 The most abundant fragment obtained in the negative mode (Figure 1b) is the odd-electron [z5 - 4H]-• ion formed through the N-CR bond cleavage between tyrosine and valine. Other oddelectron ions resulting from backbone cleavages include [z4 2H]-•, [z3 - 2H]-•, [b5 - H]-•, and [a5 - H]-•. Even-electron ions observed in the spectrum include [z3 - 3H]-, [y2 - 2H]-, and a small peak corresponding to loss of p-quinomethide from the a5 ion, [a5 - 2H - 106]-. It should be noted that it is extremely important to keep track of the hydrogen atoms in the backbone dissociation of odd-electron ions. Although the nomenclature for the dissociation of protonated peptide ions is well established,53 the assignment of fragment ions of deprotonated peptides is rather inconsistent in the literature.54-56 The situation is even more complicated for odd-electron ions. Our results demonstrate that the molecular weights of fragments produced from [M - 2H]-• ions can be shifted by the masses of one, two, three, or even four hydrogen atoms, from the molecular weights of the corresponding fragments of protonated peptides. In this study, we decided to show explicitly this hydrogen deficiency in the product ions. Small molecule losses from the precursor ion observed in the negative mode include the loss of NH3 with subsequent loss of NHdCd NH (42.0218) and NHdCdNH2• (43.0296) from the arginine side chain, the loss of p-quinomethide (C7H6O, 106.0418), and the losses of C4H9N3 (99.0796) and C3H8N3• (86.0718). Figure 1c shows the fragment distribution obtained from the 75-eV SID spectrum of the negative radical of RVYIHPF. In general, the fragmentation patterns were obtained using SORICID and SID were similar. One notable difference is that while an abundant odd-electron [x2 - H]-• (or x2-•) ion is observed in the SID spectra of the negative radical of RVYIHPF, this fragment is very small in SORI-CID spectra (Table 1). Because the appearance energy for formation of the [x2 - H]-• ion is fairly high, the differences between the SORI-CID and SID data can be attributed to the well-known discrimination against dissociation fragments formed via competitive reaction channels in slow activation methods such as SORI-CID.57,58 Specifically, once the lowest energy channel for dissociation of the precursor ion opens up, dissociation through the higher energy channel competes with further ion activation. If activation is slow compared to dissociation through the low-energy pathway, the fragment corresponding to the higher energy pathway is strongly suppressed. It should be noted that the [x2 - H]-• ion is produced from cleavage of the same C-CR bond as the dominant even-electron a5 fragment of the corresponding radical cation. Scheme 1 displays a proposed mechanism for this dissociation pathway. A similar

Scheme 1

mechanism has been used by other groups to rationalize the fragmentation behavior of peptide radical cations.33,39 It is clear that this mechanism supports the formation of a-ions if the charge remains on the N-terminus and x•-ions if the charge remains on the C-terminus. For [M - 2H]-• ions, the negative charge is most likely localized on the deprotonated C-terminus, which explains the formation of abundant C-terminal fragments. Facile formation of a-ions from the radical cation of RVYIHPF suggests that the charge in this ion is localized on the arginine residue. Indeed, the fragmentation behavior changes dramatically when arginine is replaced by histidine (see, for example, Figure 3) or another less-basic residue suggesting that the protonated arginine residue is responsible for the facile formation of a-ions in the dissociation of the radical cation of RVYIHPF. Specifically, the formation of abundant a-ions for the M+• precursor and z-ions for the [M 2H]-• precursor is not a dominant process for peptide ions that do not contain the basic arginine residue. Figure 2 shows a comparison of the MS3 ion trap spectra of +• M , [M - 2H]-•, and [M + H]2+• ions of RVYIHPF. The MS3 spectra of the M+• and [M - 2H]-• ions are quite similar to the SORI-CID spectra shown in Figures 1a and b. The major fragments of the radical cation correspond to losses of small molecules from the side chains and the formation of a-ions. Additional backbone fragments not observed in the SORI-CID spectrum of the M+• ion correspond to z4+• and z6+• ions. The major difference between the ion trap CID and SORI-CID of the [M - 2H]-• ion is the absence of the neutral losses of NH3 and the guanidino group (NHCNH) in the ion trap CID spectrum. We note that the relative abundances of the fragment ions corresponding to side-chain losses are quite different in the ion trap CID and SORI-CID experiments. This finding can be attributed to differences in the rate of ion activation in the two experiments and possible stabilization of reaction products in the ion trap experiment conducted at a higher pressure of the collision gas. In contrast with the positive and negative radicals discussed earlier, [M + H]2+• ion displays fragmentation behavior that is characteristic of both the radical cation and the protonated ion. For example, the b5+ and y2+ ions are abundant fragments of the singly and doubly protonated angiotensin III,59,60 while a-ions (a2+, a3+, a4+), z-ions (z3+•, z4+•, z5+•), and side-chain losses are characteristic of the radical cation. Most of the fragments corresponding to side-chain losses are observed as doubly charged ions. These species include the losses of p-quinomethide (106), loss of C2H5• (29), loss of COOH• (45), and loss of C4H9• (57). The only small-molecule loss observed as a singly charged fragment ion corresponds to loss of [C3H8N3 + H]+• (87). This fragment has the same mass-to-charge ratio as the fragment ion of the M+• precursor ion, suggesting that the loss of 87 Da corresponds to the radical loss of the protonated arginine side chain. The (59) Laskin, J.; Bailey, T. H.; Futrell, J. H. Int. J. Mass Spectrom. 2004, 234, 89-99. (60) Tsaprailis, G.; Nair, H.; Zhong, W.; Kuppannan, K.; Futrell, J. H.; Wysocki, V. H. Anal. Chem. 2004, 76, 2083-2094.

Figure 2. Ion trap MS3 spectra of the (a) M+• ion, (b) [M - 2H]-• ion, and (c) [M + H]2+• ions of RVYIHPF.

formation of the [C3H8N3 + H]+• fragment ion in CID of the doubly charged complex of CuII(terpy) with arginine has been previously reported.61 While there are obvious differences between the dissociation patterns of the different radical ions of angiotensin III shown in Figures 1 and 2, it is clear that some of the fragment ions are formed in both the positive and negative modes. These fragment ions, summarized in Table 1, include products formed through the loss of p-quinomethide from tyrosine, the loss of C3H8N3• from arginine, the loss of an ethyl radical from isoleucine, and the formation of the [a5 - 106]+ backbone fragment in the SORI-CID and SID experiments. The minor z4+• and [b5 - H]+• fragment ions of RVYIHPF observed in the positive mode are associated with the [z4 - 2H]-• and [b5 - 3H]-• ions observed in the negative mode. In addition, the ion trap MS3 spectra of all three radical species (Figure 2) contain a series of z-ions (z3 - z6) and an oddelectron x5 ion. Formation of these fragment ions from both positive and negative odd-electron ions can be rationalized by assuming that they are formed through charge-remote dissociation pathways. MS3 experiments indicate that the [a5 - 106]+ fragment ion is produced through consecutive fragmentation of the R-radical formed after the loss of p-quinomethide (106) from the tyrosine residue. Formation of the [a5 - 106]+ fragment ion by chargeremote mechanism most likely involves hydrogen abstraction from the β-carbon of histidine followed by fragmentation shown in Scheme 1. We also observed charge-remote fragmentation for an analogue of angiotensin III, in which the N-terminal arginine had been replaced with a histidine residue (HVYIHPF). Figure 3 displays the SORI-CID spectra of the positive and negative radicals of this peptide. The HVYIHPF peptide undergoes extensive backbone cleavages that are not limited to the formation of a-ions along with (61) Barlow, C. K.; Moran, D.; Radom, L.; McFadyen, W. D.; O’Hair, R. A. J. J. Phys. Chem. A 2006, 110, 8304-8315.

Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

6611

Figure 3. SORI-CID spectra of the (a) M+• and (b) [M - 2H]-• ion of HVYIHPF. Common noise peaks are denoted with an asterisk.

characteristic losses of small molecules from the precursor ion. Similar to the fragmentation of RVYIHPF described earlier, both the positive and negative radicals of HVYIHPF experience abundant losses of p-quinomethide, losses of C2H5•, cleavages of the C-CR bond between the proline and histidine residues, and formation of the z4+•/[z4 - 2H]-•, [b5 - H]+•/[b5 - 3H]-•, and [a5 - 106]+/[a5 - 2H - 106]- fragment ions. In addition, this peptide shows the facile loss of a radical side-chain fragment, C4H5N2• (81.0453) from the N-terminal histidine residue; this process is analogous to the loss of C3H8N3• (86.0718) from RVYIHPF. One notable difference between the two peptides is the presence of an abundant [a5 - 2H]- ion in the spectrum of the [M - 2H]-• ion of HVYIHPF (Figure 3b). Because of the absence of the very basic arginine residue in this peptide, the formation of this N-terminal fragment ion competes efficiently with the formation of the complementary C-terminal [x2 - H]-• ion. The

latter ion is a minor fragment in the SORI-CID spectra, but an abundant fragment in the SID spectra. It is interesting to compare the backbone fragmentation of the [M - 2H]-• ions observed in this study with the fragmentation behavior of radical anions produced through electron detachment from doubly deprotonated peptide ions. Zubarev and co-workers reported that the dissociation of the [M - 2H]-• ion of substance P following electron detachment resulted in the preferential cleavage of C-CR bonds and the formation of complementary oddelectron a•-ions and even-electron x-ions.35 In contrast, the backbone fragmentations of [M - 2H]-• ions reported herein are dominated by the formation of z-ions. Theoretical calculations have demonstrated that the formation of a•/x-ions is characteristic of nitrogen-centered radical anions.35,62 It has been suggested that in EDD experiments nitrogen-centered radicals are formed through electron detachment from deprotonated amide nitrogen of a peptide that does not contain acidic residues in the sequence. The absence of a•/x-ion fragments in our present spectra suggests that nitrogen-centered radicals do not contribute to the observed fragmentation. We attribute this behavior to the presence of a tyrosine residue in all of the peptides examined in this study. It has been demonstrated that the presence of the tyrosine residue facilitates the formation of radical cations31 and radical anions36 through electron transfer to trivalent metal-salen complexes. Because the phenolic hydrogen atom of tyrosine is almost as acidic as that of the C-terminal carboxyl group, it is reasonable to assume that the tyrosine side chain is the second deprotonation site for tyrosine-containing peptides. Electron transfer from the doubly deprotonated peptide to the Co(III)(salen)+ complex most likely results in formation of a distonic ion in which the charge is located on the C-terminal carboxyl group and the radical is positioned on the tyrosine side chain. It follows that the fragmentation behavior observed in this study is initiated by hydrogen atom abstraction by the tyrosyl radical resulting in the formation of C-centered radicals and their subsequent dissociation. High-

Figure 4. TFECs of charge-remote fragments of the M+• ion (top panels) and [M - 2H]-• ion (bottom panels) ions of HVYIHPF obtained at delay times of 5-ms and 1-s delay times. 6612 Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

Figure 5. SORI-CID spectra of the (a) M+• ion and (b) [M - 2H]-• ion of Tyr5-bradykinin (RPPGYSPFR). An asterisk denotes the loss of NH3 from a fragment ion.

Figure 6. SORI-CID spectra of the (a) M+• ion and (b) [M - 2H]-• ion of Tyr8-bradykinin (RPPGFSPYR).

level quantum chemistry calculations have demonstrated that C-centered radicals are generally more stable than N-centered radicals with substantial barriers for the C-N hydrogen shifts,63,64 suggesting that the relative contribution of the C-centered and N-centered radicals to the dissociation of odd-electron peptide ions may vary depending on the mode of preparation of these ions. Time- and collision-energy-resolved SID experiments provide further support for the charge-remote nature of the observed cleavages. Figure 4 provides a comparison of the fragmentation efficiency curves (FECs) obtained for several fragments of HVYIHPF obtained from time- and collision-energy-resolved SID experiments. Specifically, the FECs for the loss of p-quinomethide, the loss of C4H5N2•, and the formation of the z4+• and [a5 - 106]+ fragment ions from the M+• and [M - 2H]-• ions of HVYIHPF for reaction delays of 5 ms and 1 s are shown in the figure. While the FECs of the fragment ions of the negative radical are shifted (62) Anusiewicz, I.; Jasionowski, M.; Skurski, P.; Simons, J. J. Phys. Chem. A 2005, 109, 11332-11337. (63) Wood, G. P. F.; Moran, D.; Jacob, R.; Radom, L. J. Phys. Chem. A 2005, 109, 6318-6325. (64) Moran, D.; Jacob, R.; Wood, G. P. F.; Coote, M. L.; Davies, M. J.; O’Hair, R. A. J.; Easton, C. J.; Radom, L. Helv. Chim. Acta 2006, 89, 2254-2272.

toward higher collision energies, both the relative abundance and the kinetics of formation of the charge-remote fragment ions from the M+• and [M - 2H]-• precursors are remarkably similar. The observed similarity between the kinetics of formation of several fragments confirms that the corresponding fragmentation pathways are largely unaffected by the charge site. It follows that a large fraction of odd-electron peptide ions exist as distonic ions, in which the charge site is separated from the radical site. Figures 5 and 6 show comparisons between the SORI-CID spectra of the positive and negative radical cations of two bradykinin analogues: Tyr5-bradykinin and Tyr8-bradykinin. Clearly, the fragmentation behavior of these ions is very different from the dissociation of angiotensin analogues. Very selective backbone cleavage resulting in the formation of even-electron and oddelectron c5-ions is the dominant dissociation pathway of Tyr5bradykinin in the positive mode, while the spectrum obtained in the negative mode contains a number of abundant y-ions. Backbone fragmentation of the M+• ion of Tyr8-bradykinin results in the formation of a number of y-ions along with an abundance of even-electron and odd-electron c5- and a5-ions. The SORI-CID spectrum of the [M - 2H]-• ion of this peptide is dominated by the loss of water and other small fragments. Similarly to the dissociation trends of the angiotensin analogues discussed earlier, we observed some of the fragment ions of Tyr5bradykinin and Tyr8-bradykinin in both the positive and negative modes. These ions include fragments formed through the loss of p-quinomethide (106.0418), the loss of the entire arginine side chain (C4H9N3, 99.0796), the loss of the guanidino group (CH5N3, 59.0483), and the formation of the y7 fragment ion resulting from peptide bond cleavage between the two proline residues. In addition, the y4 - NH3 ion is a common fragment of the oddelectron ions of Tyr5-bradykinin. Our results suggest that charge-remote processes play an important role in the gas-phase dissociation of odd-electron peptide ions. We found that both side-chain cleavages and some backbone fragmentation of these ions can be attributed to charge-remote fragmentation. Particularly abundant charge-remote side-chain losses observed in this investigation include the losses of neutral tyrosine (106.0418) and arginine (99.0796) side chains, the loss of the guanidino group (59.0483), and the losses of radicals from the side chains of histidine (81.0453), arginine (86.0718), and isoleucine (29.0391). It is very likely that other side-chain losses commonly observed in the fragmentation spectra of odd-electron peptide ions result from charge-remote radical-driven bond cleavages. Our results provide solid support for the radical-driven charge-remote fragmentation mechanisms of odd-electron peptide ions proposed by other groups.33,37,39 Several studies have suggested that side-chain losses provide useful structural information for the identification of ions using ECD, ETD, or other ion activation techniques that are based on the dissociation of odd-electron peptide ions.65-67 It is interesting to compare the side-chain cleavages observed in this study with the side-chain cleavages observed in ECD. Charge reduction of doubly protonated precursor ions caused by electron capture or electron-transfer produces hydrogen-rich radical cations, [M + (65) Cooper, H. J.; Hudgins, R. R.; Hakansson, K.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2002, 13, 241-249. (66) Leymarie, N.; Costello, C. E.; O’Connor, P. B. J. Am. Chem. Soc. 2003, 125, 8949-8958. (67) Fung, Y. M. E.; Chan, T. W. D. J. Am. Soc. Mass Spectrom. 2005, 16, 15231535.

Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

6613

2H]+•. Small-molecule losses reported for ECD ions that are of interest to this study include the losses of even-electron species, e.g., the losses of C7H8O (108.058) from tyrosine, C4H6N2 (82.053) from histidine, and C4H11N3 (101.095) from arginine. We note that there is a good correspondence between the neutral losses reported for ECD, [M + 2H]+•, ions and the neutral losses from M+• and [M - 2H]-• ions. Clearly, the losses of closed-shell molecules from hydrogen-rich radical cations produced in ECD are shifted by two mass units relative to the corresponding losses observed in this study. The side-chain losses observed in the ECD spectra result in the formation of the same R-radicals that are produced through dissociation of radical cations and radical anions reported in this study. It has been proposed that R-radicals are responsible for the so-called “radical cascade” in ECD66 resulting in the multiple backbone cleavages observed in ECD spectra. It follows that there must be some similarity between the fragmentation patterns observed for different types of odd-electron peptide ions. CONCLUSIONS Comparison between the dissociation patterns obtained for the M+•, [M - 2H]-•, and [M + H]2+• peptide ions presented herein provides clear evidence that charge-remote radical-driven fragmentation processes play an important role in the dissociation of odd-electron peptide ions. Specifically, we found that side-chain losses and some backbone fragmentation of odd-electron peptide ions occur in both positive and negative modes, suggesting that the charged site is not involved in these particular fragmentation pathways. Moreover, comparison of time- and collision-energyresolved SID data obtained for these fragment ions demonstrated that both the kinetics and the relative abundance of these ions in the spectra are independent of the charge state of the precursor ion, while higher appearance energies are observed for the

6614

Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

fragments of radical anions. We conclude that these charge-remote processes involve the fragmentations of carbon-centered radicals. The lack of formation of a•/x-ions suggests that N-centered radicals do not contribute to the observed fragmentation of the odd-electron ions examined in this study. These results are particularly important because they suggest that there is a certain degree of similarity between the fragmentation behavior of the [M + 2H]+• peptide ions observed in ECD spectra, the M+• and [M - 2H]-• ions produced through gasphase fragmentation of metal-ligand complexes, and the [M 2H]-• ions observed in EDD experiments. This similarity results from charge-remote fragmentation pathways that are common for all odd-electron peptide ions. ACKNOWLEDGMENT The research described in this article was performed at the W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the U.S. Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the U.S. Department of Energy. Research at EMSL was supported by the grant from the Chemical Sciences Division, Office of Basic Energy Sciences of the US Department of Energy. I.K.C. and C.L. acknowledge participation in the PNNL Interfacial and Condensed Phase Summer Research Institute. This study was partially supported by the Hong Kong Research Grants Council, Special Administrative Region, China (Project No. HKU 7018/06P). Received for review April 18, 2007. Accepted June 25, 2007. AC070777B