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Anal. Chem. 1996, 68, 263-270
Carboxylate and Amine Terminus Directed Fragmentations in Gaseous Dipeptide Complexes with Copper(II) and Diimine Ligands Formed by Electrospray Christine L. Gatlin,† Ramesh D. Rao,† Frantisˇek Turecˇek,*,† and Toma´sˇ Vaisar‡
Department of Chemistry, Box 351700, University of Washington, Seattle, Washington 98195-1700, and Molecumetics, Ltd., Bellevue, Washington 98102
Singly and doubly charged peptide complexes with copper(II) and 2,2′-bipyridyl (bpy) are formed in the gas phase by electrospraying water-methanol solutions containing the components. Collisionally activated dissociations at low kinetic energies of singly charged complexes of the [CuII(peptide - H)(bpy)]•+ type provide information about the amino acid sequence for L-Phe-Leu, L-Leu-Phe, L-Phe-Pro, L-Pro-Phe, L-Phe-Met, L-Met-Phe, L-Ser-Phe, L-Asp-Phe, and L-His-Phe. Dissociations of doubly charged complexes of the [CuII(peptide)(bpy)]•2+ type also allow identification of the N- and C-terminal amino acid residues. Leucine and isoleucine residues are readily distinguished in L-Ala-Leu and L-Ala-Ile through dissociations of their Cu complexes. Ion dissociation mechanisms, as elucidated by deuterium labeling, are discussed. Fragmentations of gaseous peptide ions have been of much interest owing to their analytical utility for peptide sequencing.1 Gaseous peptide cations are obtained by soft ionization such as fast-atom bombardment,2 matrix-assisted laser desorption,3 or electrospray ionization (ESI)4 and made to dissociate by collisions with gaseous atoms5 or a surface6 to provide structurally significant fragment ions. Fragmentations of protonated peptide ions have been studied in detail7-9 and found to depend on, inter alia, collision energy.9,10 Sequencing of protonated peptide ions is based on charge-induced fragmentations11 that ideally occur at
random in the peptide chain and provide series of backbone fragment ions whose mass-to-charge ratios differ by the mass-tocharge ratios of one or more amino acid residues.1 Efficient protocols have been devised to sequence small peptides consisting of hydrophobic amino acids, as shown by the elegant studies of Hunt and coworkers.12 Difficulties have been encountered with peptides containing proline, lysine, arginine, and glutamine residues, which direct the protonation to the most basic sites and thus impede backbone fragmentations at other amino acid residues.13-15 The presence of internal fragments and dissociations of the amino acid side chains are some other complicating factors that occur, especially in collisionally activated dissociations (CADs) of ions with kiloelectronvolt kinetic energies.16 In contrast to the “random” fragmentations of protonated peptide ions, gas-phase peptide complexes with metal ions show some specific cleavages, depending on the metal ion and the peptide binding sites.17 For example, dissociations of peptides cationized with Li+ or Na+ commence with elimination of carbon dioxide from the carboxylate (C) terminus and proceed by cation migration along the peptide backbone.18-22 Transition metal ions23,24 form anionic complexes with multiply deprotonated peptides in the gas phase which typically provide a few sequence fragment ions on CAD.17 These previous studies used mostly fastatom bombardment to form gas-phase ions, due to complexation of the bare metal with the peptide molecule in the matrix or as a result of desorption, and employed collisions at kiloelectronvolt kinetic energies to induce ion dissociations.17-24
†
University of Washington. Molecumetics. (1) Biemann, K.; Martin, S. A. Mass Spectrom. Rev. 1987, 6, 1-76. (2) Fenselau, C.; Cotter, R. J. Chem. Rev. 1987, 87, 501-512. (3) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (4) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451. (5) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry; VCH Publishers: New York, 1988. (6) Cooks, R. G.; Ast, T.; Pradeep, T.; Wysocki, V. Acc. Chem. Res. 1994, 27, 316-323. (7) Kulik, W.; Heerma, W. Biomed. Environ. Mass Spectrom. 1988, 17, 173180. (8) Kulik, W.; Heerma, W. Biomed. Environ. Mass Spectrom. 1989, 18, 910917. (9) Boyd, R. K.; Alexander, A. J. Int. J. Mass Spectrom. Ion Processes 1989, 90, 211-240. (10) Poulter, L.; Taylor, L. C. E. Int. J. Mass Spectrom. Ion Processes 1989, 91, 183-197. (11) McLafferty, F. W.; Turecˇek, F. Interpretation of Mass Spectra, 4th ed.; University Science Books: Mill Valley, CA, 1993. ‡
(12) Cox, A. L.; Skipper, J.; Chen, Y.; Henderson, R. A.; Darrow, T. L.; Shabanowitz, J.; Engelhard, V. H.; Hunt, D. F. Science 1994, 264, 716719. (13) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111. (14) Tang, X.-J.; Thibault, P.; Boyd, R. K. Anal. Chem. 1993, 65, 2824-2833. (15) Tang, X.-J.; Boyd, R. K. Rapid. Commun. Mass Spectrom. 1994, 8, 678686. (16) Johnson, R. J.; Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Processes 1988, 86, 137-154. (17) Gross, M. L. Acc. Chem. Res. 1994, 27, 361-369. (18) Mallis, M. L.; Russell, D. H. Anal. Chem. 1986, 58, 1076-1080. (19) Renner, D.; Spiteller, G. Biomed. Environ. Mass Spectrom. 1988, 15, 7577. (20) Tang, X.; Ens, W.; Standing, K. G.; Westmore, J. B. Anal. Chem. 1988, 60, 1791-1799. (21) Teesch, L. M.; Orlando, R. C.; Adams, J. J. Am. Chem. Soc. 1991, 113, 3668-3675. (22) Zhao, H.; Adams, J. Int. J. Mass Spectrom. Ion Processes 1993, 125, 195205. (23) Hu, P.; Gross, M. L. J. Am. Chem. Soc. 1993, 115, 8821-8828. (24) Reiter, A.; Adams, J.; Zhao, H. J. Am. Chem. Soc. 1994, 116, 7827-7838.
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ESI provides a convenient tool for studying metal ion complexes in the gas phase.25-35 Previous investigations of inorganic and organometallic complexes found a correspondence between the formation of metal complexes in solution and the appearance of metal-complex ions in the gas phase following ESI.26,28-31 We have reported recently that stable gas-phase complexes can be obtained from amino acids, copper(II) salts, and auxiliary diimine ligands, such as 2,2′-bipyridyl (bpy) or phenanthroline (phen), by electrospraying methanol-water solutions containing the components.32-36 Amino acid complexes with Cu(II) and diimine or diamine ligands are known to exist in solution, with stability constants in the 1015-1017 range,37,38 and their existence in the gas phase points to their inherent stability in the absence of the solvent. The gas-phase complexes showed some novel dissociation reactions triggered by elimination of carbon dioxide, which was followed by radical-like fragmentations of the amino acid side chain.32-34 Specific fragmentations have been found to occur for the 20 essential amino acids that allowed, inter alia, distinction of the isomeric amino acids leucine and isoleucine and the isobaric amino acids lysine and glutamine.32-34 We now report on the formation of gas-phase complexes with Cu(II) and bpy of several isomeric dipeptides. Peptides are know to form stable binary and ternary complexes with Cu(II) and auxiliary ligands in both solution39 and the solid state.40 The role of the auxiliary diimine ligand in the formation of the gas-phase ions is thought to be 2-fold.33-35 First, the diimine occupies two coordination sites on Cu(II) and thus prevents formation of neutral binary complexes of the [CuII(peptide - H)2] type.32,35 Second, previous electron density calculations in the ternary complexes of amino acids with Cu and bpy showed that the diimine ligand accommodates 60-70% of the positive charge, which substantially alters the reactivity of the metal ion.33,36 In particular, dehydrogenation and metal insertions, which occur in binary complexes,17 are suppressed in the ternary complexes.32-35 We will show that (1) ESI mass spectra of ternary Cu-peptide complexes are useful for the characterization of isomeric dipeptides differing in the amino acid positions, (2) C-terminal leucine and isoleucine are readily distinguished, and (3) specific fragmentations can be induced starting from either the amino or the carboxylate terminus of the coordinated peptide. We also show that the fragmentation (25) Katta, V.; Chowdhury, S. K.; Chait, B. T. J. Am. Chem. Soc. 1990, 112, 5348-5349. (26) Blades, A. T.; Jayaweera, P.; Ikonomou, M. G.; Kebarle, P. Int. J. Mass Spectrom. Ion Processes 1990, 101, 325-336. (27) Cheng, Z. L.; Siu, K. W. M.; Guevremont, R.; Berman, S. S. J. Am. Soc. Mass Spectrom. 1992, 3, 281-288. (28) Wilson, S. R.; Wu, Y. Organometallics 1993, 12, 1478-1480. (29) Xu, X.; Nolan, S. P.; Cole, R. B. Anal. Chem. 1994, 66, 119-125. (30) Gatlin, C. L.; Turecˇek, F. Anal. Chem. 1994, 66, 712-718. (31) Gatlin, C. L.; Turecˇek, F.; Vaisar, T. Anal. Chem. 1994, 66, 3950-3958. (32) Gatlin, C. L.; Turecˇek, F.; Vaisar, T. J. Am. Chem. Soc. 1995, 117, 36373638. (33) Gatlin, C. L.; Turecˇek, F.; Vaisar, T. J. Mass Spectrom. 1995, 30, 16051616. Presented at the 7th Sanibel Conference on Mass Spectrometry, Sanibel Island, FL, January 1995. (34) Gatlin, C. L.; Turecˇek, F.; Vaisar, T. J. Mass Spectrom. 1995, 30, 16171627. (35) Gatlin, C. L.; Turecˇek, F. J. Mass Spectrom. 1995, 30, 1636-1637. (36) Gatlin, C. L.; Turecˇek, F.; Vaisar, T. J. Mass Spectrom. 1995, 30, 775-777. (37) Yamauchi, O.; Odani, A. J. Am. Chem. Soc. 1985, 107, 5938-5945. (38) Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum Press: New York, 1974; Vol. 1. (39) Sigel, H.; Naumann, C. F.; Prijs, B.; McCormick, D. B.; Falk, M. C. Inorg. Chem. 1977, 16, 790-796. (40) Lim, M. C.; Sinn, E.; Martin, B. R. Inorg. Chem. 1976, 15, 807-811.
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mechanisms of peptide-Cu ternary complexes are analogous to those elucidated for the complexes of amino acids.32-34 EXPERIMENTAL SECTION Materials. The dipeptide complexes were prepared in situ by dissolving the dipeptide (1 equiv), CuSO4‚5H2O (1 equiv,) and 2,2′-bipyridyl (bpy, 1 equiv) in 50/50 v/v aqueous methanol to achieve ∼300 µM concentration of [CuII(peptide - H)(bpy)]. Deionized water and reagent grade methanol (Baker) were used as solvents. The L-dipeptides and Asp-Phe-OCH3 (all Sigma), deuterium oxide (99.9% D), and CH3OD (99.8% D) (all Cambridge Isotope Labs) and 2,2′-bipyridyl (Aldrich) were used as received. Methyl esters Phe-Leu-OCH3 and Leu-Phe-OCH3 were synthesized by esterification with diazomethane in ether/acetonitrile and characterized by positive- and negative-ion ESI mass spectra. The β-methyl ester of Asp-Phe was synthesized by coupling N-(tertbutyloxycarbonyl)aspartic acid β-methyl ester with phenylalanine tert-butyl ester using hydroxybenzotriazole and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide in dichloromethane. The protecting groups were removed with 50% trifluoroacetic acid in dichloromethane at room temperature. The product showed one dominant peak in HPLC and gave satisfactory ESI mass and 1HNMR spectra. H/D exchange of active NH2 and amide protons in the dipeptide complexes was carried out by dissolving the peptide, anhydrous CuSO4, and bpy in a 50/50 mixture of CH3OD and D2O. The solutions were infused into the ionizer, which had been flushed with CH3OD/D2O for a few minutes. Incorporation of active deuterium atoms was better that 95% in all these measurements. Mass Spectrometry. Mass spectra were measured on a Kratos Profile HV-4 double-focusing mass spectrometer (Kratos Analytical, Ltd., Manchester, U.K.), equipped with a Kratos HVR2500 electrospray ionizer,30 and on a Fisons VG Quattro (Fisons, Manchester, U.K.) tandem quadrupole mass spectrometer equipped with a VG Fisons electrospray ionizer31 as described previously. Collision-induced dissociations41 were carried out in the first expansion region of the Kratos ionizer by increasing the potential difference between the end plate and the cage skimmers from 200 to 700 V.31 The ESI mass spectra measured on the tandem quadrupole mass spectrometer were obtained with heated dry nitrogen (90 °C) as nebulizing and drying gas. The optimized ESI source potentials for the Fisons ionizer were needle, 2.8 kV; counter electrode (“high-voltage lens”),31 50 V. Ion collisional activation in the high-pressure (0.6 Torr) region41 was carried out by ramping the voltage between the conical focus lens and the grounded second skimmer from +5 to +40 V.31 CAD of mass-selected ions used argon as the collision gas, which was admitted at a pressure to achieve 30% transmission of the precursor ion beam. The collision hexapole was floated at -5 to -25 V against the ion source corresponding to laboratory ion kinetic energies of 5-25 eV. Negative-ion ESI mass spectra were obtained with low skimmer-focus lens potentials (-10 to -12 V). RESULTS AND DISCUSSION Formation of Gaseous Cu(II)-Peptide Complexes. Electrospraying methanol/water (50/50) solutions of dipeptides, CuSO4, and bpy yields positive-ion mass spectra that show several (41) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid. Commun. Mass Spectrom. 1988, 2, 207-210.
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Figure 1. ESI mass spectra of (a) [CuII(Phe-Leu - H)bpy]•+ with the Kratos ionizer at end-plate potential of 2.2 kV and (b) [CuII(LeuPhe - H)bpy]•+ with the Fisons ionizer at skimmer potential of 10 V.
Figure 2. ESI mass spectra of the Phe-Pro complex: (a) Kratos ionizer at end-plate potential of 2.2 kV and (b) Fisons ionizer at skimmer potential of 10 V.
ion species. The peptide-containing ions appear chiefly as [peptide + H]+, [peptide + Na]+, [CuII(peptide - H)(bpy)]•+, [CuII(peptide - 2H + Na)(bpy)]•+, and [CuII(peptide)(bpy)]•2+ species. The Cu(II)-containing ions have odd numbers of electrons and are thus denoted with the •+ sign. The relative abundances in the ESI spectra of these species depend on both the peptide structure and the experimental conditions, i.e., the presence of spurious sodium ions, counterions, electrospray ionizer potentials, and the pressure regime in the differentially pumped stages of the ionizer. The spectrum of the L-Phe-Leu complex (Figure 1a) displays abundant [CuII(Phe-Leu - H)(bpy)]•+ ions at m/z 496-498, which are readily recognized through the characteristic 69.2:30.8 63Cu65Cu isotope pattern.42 In addition, the spectrum shows ions due to [bpy + H]+ (m/z 157), [bpy + Na]+ (m/z 179), [CuII(Phe-Leu)(bpy)]•2+ (m/z 248.5-249.5), [Phe-Leu + H]+ (m/z 279), [PheLeu + Na]+ (m/z 301), [CuI(bpy)2]+ (m/z 375-377), [CuII(PheLeu - 2H + Na)(bpy)]•2+ (m/z 518-520), [CuII(Phe-Leu H)(bpy)2]•+ (m/z 652-654), and a few unidentified ions (Figure 1a). The ESI mass spectrum of the [CuII(Leu-Phe - H)bpy]+ complex that was obtained with the Fisons ionizer is shown in Figure 1b. In addition to the peaks of the [CuII(Leu-Phe - H)(bpy)]•+ ions at m/z 496-498, the latter spectrum shows abundant [CuII(Leu-Phe - 2H + Na)(bpy)]•+ (m/z 518-520) due to the presence of spurious Na+ ions, which were found to originate from the glass vials used to store the solutions. The sodiated ions can be suppressed completely by preparing and storing the solutions in polypropylene vials. The ESI spectra of the Phe-Pro, Pro-Phe, Phe-Met, Met-Phe, Ala-Leu, and Ala-Ile complexes show abundant doubly charged ions of the [CuII(peptide)(bpy)]•2+ type whose relative abundances
depend on experimental conditions, in particular, the pressure and potentials in the ESI ionizer. For example, ionization in the Kratos ionizer at >10 Torr gives [CuII(Phe-Pro - H)(bpy)]•+ at m/z 480482 as the most abundant ions (100%) and [CuII(Phe-Pro)(bpy)]•2+ at m/z 240.5-241.5 of only 13% relative abundance (Figure 2a). Ionization in the Fisons ionizer at 0.6 Torr yields [CuII(Phe-Pro)(bpy)]•2+ of 90% relative abundance, in addition to the singly charged [CuII(Phe-Pro - H)(bpy)]•+ ions (100%, Figure 2b). Similar trends in the formation of singly and doubly charged ions have been observed for CuII(bpy) complexes of Pro-Phe, MetPhe, Phe-Met, Ala-Leu, and Ala-Ile. Apparently, collisions with the background methanol and water molecules of the gas-phase peptide ions affect the distribution of the charged species by proton transfer from the peptide ligand to the gaseous solvent molecules. This is analogous to collisional deprotonation of multiply charged protein ions in electrospray.41,43 From a practical point of view, the observed sensitivity to experimental conditions means that ESI spectra of the peptide complexes obtained with different ESI ionizers or in different pressure regimes should be compared with caution. The ESI spectra of the His-Phe, Asp-Phe, and Ser-Phe complexes show abundant singly charged complexes, whereas the doubly charged [CuII(peptide)(bpy)]•2+ ions are weak (10 Torr),33,36 decarboxylation was, in general, the major dissociation, and a few structurally significant ions were observed or distinguished from solvent adduct ions.36 Second, CAD in the Fisons ionizer of nonselected ions was used to prepare fragment ions to be then selected by mass and submitted to further CAD. In this way, MS/MS/MS is simulated without mass selection of the precursor ion but with mass selection of the intermediate ion. Third, CAD of mass-selected ions were monitored under welldefined conditions of pressure and kinetic energy. The latter spectra are discussed throughout. The presence of copper in the product ions was determined from the mass shifts in the CAD spectra of mass-selected 63Cu and 65Cu isotopomers. The CAD spectrum of the [CuII(Phe-Leu - H)(bpy)]•+ ion (m/z 496 for 63Cu) shows dominant formation of [Cu(NH2)(bpy)]+ at m/z 235 and [Cu(bpy)]+ at m/z 219 (Figure 3a). In addition, decarboxylation gives rise to the ion at m/z 452, which further loses C3H7• to form the ion at m/z 409. The latter fragmentation sequence is typical for Cu-leucine complexes, whereby the C3H7•
Figure 3. CAD spectra of (a) [CuII(Phe-Leu - H)(bpy]•+ and (b) [CuII(Leu-Phe - H)(bpy)]•+ at ELAB ) 15 eV.
radical is eliminated from the leucine side chain by a radical-like β-fission.32,44 The presence of Phe is indicated by the loss of the benzyl group to yield the ion at m/z 405. A sequence ion is observed at m/z 338, which corresponds to a [Cu(C6H5C2H4N)(bpy)]+ complex from the N-terminal Phe. Formation of the latter ion is analogous to an a-type cleavage,13,45 with concomitant transfer of the Cu(bpy) residue to the N-terminus and reverse (44) Beckwith, A. L. J. Tetrahedron 1981, 37, 3073-3100.
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+
Scheme 2
transfer of a proton onto the neutral fragment (Scheme 1). These assignments were corroborated by H/D exchange of the NH and OH protons, resulting in >95% incorporation of three deuterium atoms in the gaseous ion.46 The sequence a-type ion incorporates 1-2 deuterium atoms, indicating intramolecular H/D exchange in the fragmentation. The ion is best represented as a mixture of imine and enamine structures (Scheme 1). The [CuII(Leu-Phe - H)(bpy)]+ ion gives an abundant sequence a-type ion at m/z 304 (Figure 3b), which corresponds to a Cu(bpy)-imine complex from the leucine residue (Scheme 2). Deuterium labeling in Leu-Phe shows clean exchange of the labile protons to give [CuII(Leu-Phe-d3 - D)(bpy)]+ ions at m/z 499501.46 The CAD spectrum of the 63Cu isotopomer (m/z 499) shows mass shifts of m/z 452 f 455, m/z 396 f 399, m/z 296 f 299, m/z 240 f 243, and retention of m/z 435 due to elimination of ND3. The sequence a-type ion, appearing at m/z 305, shows retention of one deuterium atom from the amino group, consistent with the proposed imine structure. The [CuII(Phe-Pro - H)(bpy)]•+ and [CuII(Pro-Phe - H)(bpy)]•+ complexes give distinct CAD spectra, which show fragmentations typical of the amino acid residues (Figure 4). Loss of the benzyl group (m/z 389) and the formation of the sequence a-type ion (m/z 338) characterize the Phe-Pro isomer (Figure 4a). On H/D exchange of the active hydrogen atoms in Phe-Pro, the sequence a-type ion at m/z 338 shows a distribution of D0, D1, and D2 species at m/z 338, 339, and 340 in a 1:3:1.5 ratio. Hence, both the nitrogen- and carbon-bound hydrogen atoms are transferred in the formation of the a-type ion. The [CuII(Pro-Phe H)(bpy)]•+ complex gives a dominant sequence a-type ion at m/z 288 (Table 1), which corresponds to the [Cu(∆1-pyrroline)(bpy)]+ complex.33 Deuterium labeling by H/D exchange of the Pro-Phe active hydrogen atoms results in the sequence a-type ion consisting of D0 and D1 species in a 2.2:1 ratio. This indicates prevalent formation of the ∆1-pyrroline (imine) complex from the proline residue.33 Phe-Met and Met-Phe are readily distinguished through the CAD spectra of their [CuII(Phe-Met - H)(bpy)]•+ and [CuII(MetPhe - H)(bpy)]•+ complexes (m/z 514 for 63Cu, Figure 5). In particular, the Phe-Met isomer gives an a-type sequence ion at m/z 338 and a dominant peak of [Cu(NH2)(bpy)]+ at m/z 235, which are typical for the N-terminal Phe residue (Figure 5a). The (45) Roepstorff, P.; Fohlman, J. Biomed. Environ. Mass Spectrom. 1984, 11, 601. (46) These spectra are available as supporting information.
Figure 4. CAD spectra of (a) [CuII(Phe-Pro - H)(bpy]•+ and (b) [CuII(Pro-Phe - H)(bpy)]•+ at ELAB ) 15 eV. Table 1. Relative Intensities of Sequence a-Type Ions from Dipeptide Cu Complexes a-type ion
dipeptide
precursor ion m/za
m/za
rel intensb
Leu-Phe Phe-Leu Met-Phe Phe-Met Pro-Phe Phe-Pro Ser-Phe Asp-Phe His-Phe Ala-Leu Ala-Ile
496 496 514 514 480 480 470 498 520 420 420
304 338 322 338 288 338 278 306 328 262 262
14 3.0 22 3.3 53 2.4 6.7 10 27 18 23
a For 63Cu isotopes. b Relative to the sum of CAD fragment ion intensities at ELAB ) 15 eV.
[CuII(Met-Phe - H)(bpy)]•+ complex produces ions due to the cleavage of the methionine side chain in combination with decarboxylation. The dominating fragments are [Cu(CH3S)(bpy)]•+ at m/z 266 and the sequence a-type ion at m/z 322 (Figure 5b). Further CAD at 10 eV ion kinetic energy of the latter ion leads to the formation of [Cu(bpy)]+ at m/z 219 and [Cu(CH3SCH2CH2CHdNH)]+ at m/z 166 in a 9:1 ratio, indicating a stronger binding to Cu(I) of bpy compared with the sulfur-containing ligand. The CAD spectrum of the deuterium-labeled Met-Phe-d3 complex is consistent with the suggested dissociation pathways.46 In particular, the sequence a-type ion shows a very clean mass shift by 1 unit, indicating retention of a single amine deuteron, in keeping with the proposed imine structure for the ligand. Structure information is also obtained from the CAD spectra of CuII(bpy) complexes of Ser-Phe, Asp-Phe, and His-Phe (Table 1). The [CuII(Ser-Phe - H)(bpy)]•+ ion (m/z 470 for 63Cu) undergoes elimination of CH2O, which is typical for serine-
+
Figure 5. CAD spectra of (a) [CuII(Phe-Met - H)(bpy]•+ and (b) [CuII(Met-Phe - H)(bpy)]•+ at ELAB ) 15 eV.
containing complexes.22,23,33 The sequence a-type ion appears at m/z 278 (Table 1). On H/D exchange, the latter ion undergoes a clean mass shift to m/z 280, which indicates retention of two active deuterium atoms in the DOCH2CHdND ligand from the serine residue.46 The sequence a-type ion from [CuII(Asp-Phe - H)(bpy)]•+ appears at m/z 306 (Table 1). On H/D exchange it shows retention of two and three deuterium atoms in a 2.2:1 ratio, which suggests a more favorable formation of an imino ligand from the aspartate residue. It is noteworthy that CAD of the [CuII(AspPhe - H)(bpy)]•+ complex is less efficient than CAD of the other dipeptide complexes and results in the dominant formation of [Cu(bpy)]+ due to loss of the whole peptide ligand. Owing to the presence of two carboxylic groups in Asp-Phe, it is conceivable that coordination to Cu(II) can occur by either the terminal or the sidechain β-carboxylic group to form a mixture of complexes that may have different reactivities on CAD. We pursued this question in more detail by investigating the ESI/CAD spectra of Cu complexes of Asp-Phe monomethyl esters in which either the C-terminal or the aspartate β-carboxylate group was blocked. Both methyl esters form singly charged gas-phase complexes with Cu(bpy), which appear at m/z 512-514. Upon CAD at ELAB ) 20 eV, the m/z 512 ion from the C-terminus methylated dipeptide eliminates ammonia and COOCH3, or the whole peptide residue, to give [Cu(bpy)]+. However, the predominant dissociation is the formation of an a-type fragment at m/z 306, which amounts to 46% of the total CAD ion intensities (∑CAD). This ion is analogous to, but not necessarily identical with, the sequence ion from [CuII(Asp-Phe - H)(bpy)].•+ CAD of the m/z 512 ion from the aspartate-methylated dipeptide results in elimination of CO2 from the C-terminus and formation of abundant fragment ions at m/z 235 and 219. An a-type sequence ion appears at m/z 320 (20% ∑CAD), and its mass shift by 14 u confirms the presence of the
+
Figure 6. CAD spectra of (a) [CuII(Ala-Leu - H)(bpy)]•+ and (b) [CuII(Ala-Ile - H)(bpy)]•+ at ELAB ) 15 eV.
aspartate carboxymethyl group. These results show clearly that Asp-Phe can coordinate to Cu(II) by either carboxylic group. It is noteworthy that CAD of the binuclear complex, {[CuII(bpy)]2(AspPhe - 2H)}2+, in which both carboxylate groups are presumably coordinated, gives mostly [Cu(bpy)]+ and a singly charged fragment at m/z 297, the latter due to elimination of [Cu(bpy)]+, CO2, and bpy. The [CuII(His-Phe - H)(bpy)]+ complex undergoes decarboxylation on CAD and forms a sequence a-type ion at m/z 328 (Table 1). On H/D exchange, the a-type ion shows D2 and D3 species in a 1:1 ratio, which indicates the formation of both the imino and enamino isomers of the dehydrohistamine ligand.33 The specific fragmentations of the gas-phase Cu-dipeptide complexes allow one to identify the amino acid residue at the C-terminus. This is demonstrated with isomeric dipeptides AlaLeu and Ala-Ile, which both form singly and doubly charged Cu complexes, which appear at m/z 420 and 210.5 for the 63Cu isotopomers, respectively. While the ESI spectra of the isomeric dipeptide complexes are very similar, the CAD spectra of the m/z 420 ions from [CuII(Ala-Leu - H)(bpy)]•+ and [CuII(Ala-Ile - H)(bpy)]•+ differ. Following decarboxylation, the Ala-Leu complex eliminates a C3H7• radical from the leucine residue to form the ion at m/z 333 (Figure 6a). The sequence a-type ion, corresponding to Ala, appears at m/z 262. The Ala-Ile complex undergoes decarboxylation (m/z 376, Figure 6b), followed by losses of CH3• (m/z 361) and C2H5• (m/z 347) and formation of a sequence a-type ion at m/z 262. The side chain fragmentations in the singly charged ions thus clearly distinguish C-terminal leucine from isoleucine in the dipeptides. Also noteworthy is the more facile loss of the isopropyl radical from the Ala-Leu isomer. Collisionally Activated Dissociations of Doubly Charged Ions. Further structure information is obtained from the CAD spectra of doubly charged Cu-peptide ternary complexes, which show efficient dissociations at collision energies as low as 5 eV.
+
Figure 7. CAD spectra of (a) [CuII(Ala-Leu)(bpy)]•2+ and (b) [CuII(Ala-Ile)(bpy)]•2+ at ELAB ) 5 eV.
Scheme 3
Doubly charged complexes of Phe-Pro, Pro-Phe, Phe-Met, MetPhe, Ala-Leu, and Ala-Ile undergo a few simple dissociations, as summarized in Scheme 3 and illustrated for Ala-Leu and Ala-Ile (Figure 7). The [CuII(Ala-Leu)(bpy)]•2+ ions (m/z 210.5) eliminate the alanine residue to form the singly charged [CuII(Leu - H)(bpy)]•+ ion at m/z 349 (Figure 7a). The latter further dissociates by successive losses of CO2 and C3H7• to give the ions at m/z 305 and 262, respectively. Likewise, CAD of [CuII(Ala-Ile)(bpy)]•2+ produces the [CuII(Ile - H)(bpy)]•+ ion at m/z 349, which eliminates successively CO2 and C2H5 (Figure 7b) to give rise to ions at m/z 305 and 276, respectively. Alanine immonium ions, CH3CHdNH2+ at m/z 44, are observed for both the Ala-Leu and Ala-Ile complexes and characterize the N-terminal amino acid. These ion series thus provide complete information on the amino acid sequence, structure of the C-terminal amino acid, and the site of Cu(bpy) attachment in the doubly charged complex. It should be noted in this context that CADs of protonated dipeptides
+
also provide sequence-specific ions, e.g., immonium (a) ions characterizing the N-terminus and y′′ ions characterizing the C-terminus.7,8 However, distinction of the Leu and Ile residues at the C-terminus is difficult if not impossible through CAD spectra of protonated peptides.7,8 The dissociations of isomeric doubly charged complexes [CuII(Phe-Pro)(bpy)]•2+ and [CuII(Phe-Pro)(bpy)]•2+ follow a similar general pattern.46 Loss of the N-terminal amino acid residue produces [CuII(Pro - H)(bpy)]•+ (m/z 333) and [CuII(Phe - H)(bpy)]•+ (m/z 383) from the Phe-Pro and Pro-Phe doubly charged complexes, respectively. These ions undergo further dissociations that are specific for the corresponding amino acid complexes, e.g., decarboxylation and side chain cleavages.32-34 The N-terminal amino acids in the dipeptide complexes give rise to immonium ions, m/z 120 and 70 for Phe-Pro and Pro-Phe, respectively.46 The doubly charged ion, [CuII(Met-Phe)(bpy)]•2+ at m/z 257.5, eliminates CH3SH to give a doubly charged fragment at m/z 234.46 The methionine immonium ion, CH3SCH2CH2CHdNH2+, appears at m/z 104 to characterize the N-terminal amino acid. The C-terminus manifests itself through the [Cu(Phe - H)(bpy)]•+ ion and its dissociation products.32,33 The isomeric [CuII(Phe-Met)(bpy)]•2+ complex gives rise to an abundant phenylethylimmonium ion at m/z 120.46 The copper-containing singly charged ions comprise [CuII(Met - H)(bpy)]•+ at m/z 367 and its known fragments at m/z 323, 308, 280, 277, 266, 265, 262, and 219,34 as corroborated by the mass shifts in the CAD spectrum of the 65Cu isotopomer. Both the amino and the carboxylate termini are thus characterized through the CAD spectrum of the doubly charged Cu complex. Ion Structures and Dissociation Mechanisms. The dissociations of the [CuII(peptide - H)(bpy)]•+ ions show some common features that are indicative of the metal binding in the complex. Coordination by the carboxyl group has been presumed for binary anionic complexes of Ni2+ and Co2+ with multiply deprotonated peptides in solution47 and the gas phase23,24 and confirmed in the solid state by an X-ray structure.40 By contrast, ternary [CuII(peptide - H)(bpy)]•+ complexes in solution have been depicted with deprotonation occurring at the amide nitrogen atom, such that the amide oxygen and the N-terminal amino group provided two binding sites for the Cu(II) ion.39 The gas-phase data point to peptide coordination by the carboxylate group, in close analogy with gas-phase Cu(II) complexes of a variety of carboxylic acids (acetic, propionic, lactic, and R- and β-amino acids), which lack a peptide bond and must deprotonate at the carboxylic group.31-35 The gas-phase dissociations of these carboxylate Cu(II) complexes proceed by decarboxylation, similar to those of the Cu(II)-peptide ions.31-35 A definite proof of carboxylate coordination in the peptide complexes is provided by the ESI spectra of methyl esters, Phe-Leu-OCH3, Leu-Phe-OCH3, Pro-Leu-OCH3, and Leu-Pro-OCH3, in which deprotonation of the carboxylate terminus is blocked. The ESI spectra of these methyl esters in the presence of Cu(II) and bpy show intense protonated dipeptide molecules, (M + H)+, e.g., m/z 293 and 243 for PheLeu-OCH3 and Pro-Leu-OCH3, respectively, but
