Anal. Chem. 2005, 77, 1796-1806
Characterization of Dipeptide Isomers by Tandem Mass Spectrometry of Their Mono- versus Dilithiated Complexes Francesco Pingitore and Chrys Wesdemiotis*
Department of Chemistry, The University of Akron, Akron, Ohio 44325-3601 The Li+ complexes of the isomeric dipeptide pairs PheGly/ GlyPhe, PheAla/AlaPhe, and TrpAla/AlaTrp, namely, [Pep + Li]+, and of the corresponding lithium carboxylates, namely, [Pep - H + 2Li]+, are produced in the gas phase by desorption ionization, and their unimolecular chemistry is probed by tandem mass spectrometry experiments at various activation conditions. At low internal energies, monolithiated isomers dissociate to the same products, formed through a mixed anhydride intermediate in which the sequence information is lost. Isomerization to the mixed anhydride is less competitive at higher internal energies, which start promoting sequence-specific fragmentations. On the other hand, dilithiated isomers (they contain a permanent COO-Li+ salt bridge) do not rearrange to an anhydride and give rise to substantially different fragmentation patterns; structurally diagnostic c1- and y1-type fragments are observed at all internal energies, allowing for unequivocal sequence assignment. The mono- and dilithiated peptides undergo loss of their aromatic side chain to form distonic radical ions carrying Li+ charge(s) and one unpaired electron at an r-C atom of the peptide backbone. The yield of such metal-bound peptide radicals is particularly high from the dilithiated complexes, [Pep - H + 2Li]+. Upon activation, the Li+ ions become mobile and can be shuttled to the various basic sites of the dipeptides, where they may initiate backbone fragmentation or the elimination of small neutral molecules. The characterization of peptide sequences by tandem mass spectrometry (MS/MS) usually employs protonated peptides1-4 which can readily be formed by desorption or spray ionization techniques, such as fast atom bombardment (FAB),5 matrixassisted laser desorption ionization (MALDI),6,7 and electrospray * Corresponding author. Phone: (330) 972-7699. Fax: (330) 972-7370. E-mail:
[email protected]. (1) Biemann, K. Methods Enzymol. 1990, 193, 455-479. (2) Papayannopoulos, I. A. Mass Spectrom. Rev. 1995, 14, 49-73. (3) Kinter, M.; Sherman, N. E. Protein Sequencing and Identification Using Tandem Mass Spectrometry; Wiley-Interscience: New York, 2000. (4) Siuzdak, G. The Expanding Role of Mass Spectrometry in Biotechnology; MCC Press: San Diego, 2003. (5) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. Nature 1981, 293, 270-275. (6) Yoshida, T.; Tanaka, K.; Ido, Y.; Akita, Y.; Yoshida, Y. Shitsutyo Bunseki 1988, 36, 59-69.
1796 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005
ionization (ESI).8 The [Pep + H]+ (or [Pep + 2H]2+ in ESI) ions are mass-selected and subsequently fragmented by collisionally activated dissociation (CAD). This process leads to cleavages along the peptide backbone and in the side chains. Random dissociation at the amide bonds in the backbone generates a series of fragments containing either the N terminus (bn ions) or the C terminus (yn ions), on the basis of which the original peptide sequence can be reconstructed.1-4 Protonated peptides do not always yield a sufficient number of backbone fragments for definitive sequence elucidation.3 For this reason, several research groups have examined metalated peptides as precursor ions.9-31 The latter studies provided alternative means to deduce peptide (7) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (8) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F. Science 1989, 246, 6471. (9) Mallis, L. M.; Russell, D. H. Anal. Chem. 1986, 58, 1076-1080. (10) Russell, D. H.; McGlohon, E. S.; Mallis, L. M. Anal. Chem. 1988, 60, 18181824. (11) Renner, G.; Spiteller, G. Biomed. Environ. Mass Spectrom. 1988, 15, 7577. (12) Tang, X.; Ens, W.; Standing, K. G.; Westmore, J. B. Anal. Chem. 1988, 60, 1791-1799. (13) Grese, R. P.; Cerny, R. L.; Gross, M. L. J. Am. Chem. Soc. 1989, 111, 28352842. (14) Leary, J. A.; Williams, T. D.; Bott, G. Rapid Commun. Mass Spectrom. 1989, 3, 192-196. (15) Leary, J. A.; Zhou, Z.; Ogden, S. A.; Williams, T. D. J. Am. Soc. Mass Spectrom. 1990, 1, 473-480. (16) Grese, R. P.; Gross, M. L. J. Am. Chem. Soc. 1990, 112, 5098-5104. (17) Teesch, L. M.; Adams, J. J. Am. Chem. Soc. 1991, 113, 812-820. (18) Teesch, L. M.; Orlando, R. C.; Adams, J. J. Am. Chem. Soc. 1991, 113, 3668-3675. (19) Hu, P.; Gross, M. L. J. Am. Chem. Soc. 1992, 114, 9163-9160. (20) Hu, P.; Gross, M. L. J. Am. Chem. Soc. 1993, 115, 8821-8828. (21) Loo, J. A.; Hu, P.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1994, 5, 959965. (22) Wang, J.; Guevremont, R.; Siu, M. K. W. Eur. Mass Spectrom. 1995, 1, 171-181. (23) Hu, P.; Loo, J. A. J. Am. Chem. Soc. 1995, 117, 11314-11319. (24) Summerfield, S. G.; Dale, V. C. M.; Despeyroux, D. D.; Jennings, K. R. Eur. Mass Spectrom. 1995, 1, 183-194. (25) Nemirovskiy, O. V.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1996, 7, 977980. (26) Nemirovskiy, O. V.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1998, 9, 10201028. (27) Lee, S. W.; Kim, H. S.; Beauchamp, J. L. J. Am. Chem. Soc. 1998, 120, 3188-3195. (28) Lin, T.; Glish, G. L. Anal. Chem. 1998, 70, 5162-5165. (29) Cerda, B. A.; Cornett, L.; Wesdemiotis, C. Int. J. Mass Spectrom. 1999, 193, 205-226. (30) Barr, J. M.; Van Stipdonk, M. J. Rapid Commun. Mass Spectrom. 2002, 16, 566-578. (31) Kish, M. M.; Wesdemiotis, C. Int. J. Mass Spectrom. 2003, 227, 191-203. 10.1021/ac048469q CCC: $30.25
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sequences as well as new insight about the possible binding sites of metal ions on peptides. A particularly useful collision-induced fragmentation of the alkali metal ion complexes of peptides is the elimination of the C-terminal amino acid residue to yield truncated peptide-metal ion complexes, referred to as [bn + OH + M]+ cations (M ) Li, Na, K).9-18,28-32 For example, CAD of [XxxYyyZzz + M]+ produces the truncated ion [XxxYyy + M]+, a [b2 + OH + M]+ fragment ion. This rearrangement dissociation is most pronounced with the Li+ and Na+ complexes of peptides and, in a trap mass spectrometer, can be repeated to obtain the C-terminal peptide sequence. 28 A number of studies have interrogated the mechanism of [bn + OH + M]+ formation for alkali-metalated peptides.11-13,16-18,32,33 Such fragments are only observed if the peptide contains an underivatized C terminus (i.e., COOH).13,18 From isomeric dipeptides (XxxYyy vs YyyXxx), both [Xxx + M]+ and [Yyy + M]+ fragments are formed with similar yields independent of sequence.16,33 These facts were recently reconciled by an experimental and computational investigation of Gronert et al.,33 who showed that the C-terminal residue is lost through an anhydride intermediate; with isomeric dipeptides, the same intermediate is formed from either sequence, explaining the lack of specificity mentioned above. A similar mechanism involving a mixed anhydride was proposed by Farrugia and O’Hair to explain the identical tandem mass spectra of isomeric arginine-containing protonated dipeptides.34 Here, we investigate the fragmentation patterns of the Li+ complexes of PheGly/GlyPhe and other isomeric dipeptides as a function of internal energy. The major objectives are to assess the energetics of [b1 + OH + Li]+ formation and evaluate the competitive fragmentations taking place, especially those leading to radical cations (metal-ion bound radicals) which are of special interest to concurrent studies in our group. The influence of salt bridges on fragmentation is also addressed by the co-investigation of the dilithiated complexes [Pep - H + 2Li]+ that contain permanent COO-Li+ ion pairs. As will be shown, the dilithiated species permit an unequivocal characterization of dipeptide sequences via MS/MS, which may be impossible with protonated or mono-metalated precursor ions because of the aforementioned isomerization through a mixed anhydride. EXPERIMENTAL SECTION Tandem Mass Spectrometry (MS/MS) Experiments. The experiments were conducted on a Micromass AutoSpec-Q tandem mass spectrometer of EBhQ geometry (E, electric sector; B, magnetic sector; h, RF-only hexapole; Q, quadrupole mass filter).35,36 Mono- and dilithiated peptide ions ([Pep + Li]+ and [Pep - H + 2Li]+, respectively) were formed by FAB ionization, using 12 keV Cs+ ions as bombarding particles and R-thioglycerol as the matrix. The ions were accelerated to 8 keV and massselected by the EB sectors for measurement of their metastable (32) Anbalagan, V.; Silva, A. T. M.; Rajagopalachary, S.; Bulleigh, K.; Talaty, E. R.; Van Stipdonk, M. J. J. Mass Spectrom. 2004, 39, 495-504. (33) Feng, W. Y.; Gronert, S.; Fletcher, K. A.; Warres, A.; Lebrilla, C. B. Int. J. Mass Spectrom. 2003, 222, 117-134. (34) Farrugia, J. M.; O’Hair, R. A. J. Int. J. Mass Spectrom. 2003, 222, 229-242. (35) Polce, M. J.; Cordero, M. M.; Wesdemiotis, C.; Bott, P. A. Int. J. Mass Spectrom. Ion Processes 1992, 113, 35-58. (36) Pingitore, F.; Polce, M. J.; Wang, P.; Wesdemiotis, C.; Paizs, B. J. Am. Soc. Mass Spectrom. 2004, 15, 1025-1038.
ion (MI) and collisionally activated dissociation (CAD) tandem mass spectra at high kinetic energy in the field-free region (FFR) between EB and the subsequent electric sector. The product ions from these reactions were mass-analyzed by scanning the second electric sector. In CAD mode, one of the collision cells situated in the FFR was pressurized with argon to effect 80% transmittance of the precursor beam. In triple-stage (MS3) experiments, a specific fragment from metastable precursor ions dissociating in the field-free region in front of the first electric sector was transmitted through EB by proper adjustment of the E and B fields, and the corresponding high-energy CAD spectrum was acquired using the above-mentioned collision cell. Low-energy CAD spectra (MS2 mode) were obtained by transmitting the precursor ions through all three sectors (EBE), decelerating them to a laboratory-frame kinetic energy (Elab) of
