Thomas W. Chung

Backbone and side-chain specific dissociations of z ions from non-tryptic peptides

Backbone z-type fragment ions formed by electron-transfer dissociation (ETD) of doubly protonated peptides AAHAL, AHDAL, and AHADL were subjected to collisional activation and their dissociation products were studied by ETD-CID-MS3 and MS4. Electron structure theory calculations were performed to elucidate ion structures and reaction mechanisms. All z ions showed competitive eliminations of C3H7 and C4H8 from the C-terminal Leu side chain. The energetics and kinetics of these dissociations were studied computationally for the z4 ion from AAHAL, and optimized structures are reported for several intermediates and transition states. RRKM calculations on the combined B3LYP and PMP2/6-311++G(2d,p) potential energy surface provided unimolecular rate constants that closely reproduced the experimental branching ratios for C3H7 and C4H8 eliminations. Mechanisms were also studied for the loss of CO2 from z ions generated by ETD of AHDAL and AHADL and for a specific radical-induced Asp-Cα-CO backbone cleavage. CID of the z ions under study did not produce any fragment ions that would indicate cascade backbone dissociations triggered by the radical sites. In contrast, the majority of backbone dissociations occurred at bonds that were remote from the radical sites (spin-remote dissociations) and were triggered by proton migrations that were analogous to those considered for standard peptide ion fragmentations.

Electron-Capture and -Transfer Dissociation of Peptides Tagged with Tunable Fixed-Charge Groups: Structures and Dissociation Energetics

Pyridiniummethylcarbonyl moieties that were previously designed on the basis of electronic structure analysis are now utilized as fixed-charge tags with tunable electronic properties to be used for N-terminal peptide derivatization and sequencing by electron-transfer dissociation. Dipeptides AK and KA were derivatized at the peptide N-terminus with 4-dimethylaminopyridinium-N-acetyl (DMAP-ac) and pyridinium-N-acetyl (pyrid-ac) tags of increasing intrinsic recombination energies. Upon the capture of a free electron or electron transfer from fluoranthene anions, (DMAP-ac-AK+H)2+, (DMAP-ac-KA+H)2+, (pyrid-ac-AK+H)2+ and (pyrid-ac-KA+H)2+ ions, as well as underivatized (AK+2H)2+, completely dissociated. The fixed-charge tags steered the dissociation upon electron transfer to form abundant backbone N–Cα bond cleavages, whereas the underivatized peptide mainly underwent H-atom and side-chain losses. Precursor ion structures for the tagged peptides were analyzed by an exhaustive conformational search combined with B3LYP/6-31+G(d,p) geometry optimization and single-point energy calculations in order to select the global energy minima. Structures, relative energies, transition states, ion–molecule complexes, and dissociation products were identified for several charge-reduced species from the tagged peptides. The electronic properties of the charge tags and their interactions with the peptide moieties are discussed. Electrospray ionization and electron-transfer dissociation of larger peptides are illustrated with a DMAP-tagged pentapeptide.

Tunable Charge Tags for Electron-Based Methods of Peptide Sequencing: Design and Applications

Charge tags using basic auxiliary functional groups 6-aminoquinolinylcarboxamido, 4-aminopyrimidyl-1-methylcarboxamido, 2-aminobenzoimidazolyl-1-methylcarboxamido, and the fixed-charge 4-(dimethylamino)pyridyl-1-carboxamido moiety are evaluated as to their properties in electron transfer dissociation mass spectra of arginine C-terminated peptides. The neutral tags have proton affinities that are competitive with those of amino acid residues in peptides. Charge reduction by electron transfer from fluoranthene anion-radicals results in peptide backbone dissociations that improve sequence coverage by providing extensive series of N-terminal c-type fragments without impeding the formation of C-terminal z fragments. Comparison of ETD mass spectra of free and tagged peptides allows one to resolve ambiguities in fragment ion assignment through mass shifts of c ions. Simple chemical procedures are reported for N-terminal tagging of Arg-containing tryptic peptides.

Electronic properties of charge-tagged peptides upon electron capture

We report a computational study of Ala–Lys (AK) and Lys–Ala (KA) dipeptide ions furnished with fixed-charged pyridinium groups that were attached by amide linkers to the N-terminal amino groups. Cation–radicals from one-electron reduction of the doubly charged AK and KA peptide conjugates showed various extents of unpaired electron density being delocalized between the pyridine and peptide moieties. The delocalization depended on the local recombination energies (REloc) of the charged groups. The REloc of the pyridine moieties were modified by introducing electron-donating substituents (CH3, OCH3, and N(CH3)2). The REloc of the peptide moieties were found to depend on the peptide conformation and internal solvation of the Lys ammonium groups. Substantial electron delocalization was found for combinations of pyridine substituents and peptide conformers with closely matched REloc, such as 4-dimethylaminopyridine and internally solvated Lys ammonium or unsubstituted pyridine and free (unsolvated) Lys ammonium. The dissociation (ΔHdiss) and transition state energies (ETS) for the loss of the pyridine ring from the conjugates were found to be ΔHdiss = 34–36 kJ mol−1 and ETS = 67–69 kJ mol−1 for the unsubstituted pyridine moieties, but did not depend much on the peptide sequence.

Perturbing Peptide Cation-Radical Electronic States by Thioxoamide Groups: Formation, Dissociations, and Energetics of Thioxopeptide Cation-Radicals

Thioxodipeptides Gly-thio-Lys (GtK), Ala-thio-Lys (AtK), and Ala-thio-Arg (AtR) in which the amide group has been modified to a thioxoamide were made into dications by electrospray ionization and converted to cation-radicals, (GtK + 2H)(+•), (AtK + 2H)(+•), and (AtR + 2H)(+•), by electron transfer dissociation (ETD) tandem mass spectrometry using fluoranthene anion-radical as an electron donor. The common and dominant dissociation of these cation-radicals was the loss of a hydrogen atom. The dissociation products were characterized by collision-induced dissociation (CID) multistage tandem mass spectrometry up to CID-MS(5). The ground electronic states of several (GtK + 2H)(+•), (AtK + 2H)(+•), and (AtR + 2H)(+•) conformers were explored by extensive ab initio and density functional theory calculations of the potential energy surface. In silico electron transfer to the precursor dications, (GtK + 2H)(2+), (AtK + 2H)(2+), and (AtR + 2H)(2+), formed zwitterionic intermediates containing thioenol anion-radical and ammonium cation groups that were local energy minima on the potential energy surface of the ground electronic state. The zwitterions underwent facile isomerization by N-terminal ammonium proton migration to the thioenol anion-radical group forming aminothioketyl intermediates. Combined potential energy mapping and RRKM calculations of dissociation rate constants identified N-C(α) bond cleavages as the most favorable dissociation pathways, in a stark contrast to the experimental results. This discrepancy is interpreted as being due to the population upon electron transfer of low-lying excited electronic states that promote loss of hydrogen atoms. For (GtK + 2H)(+•), these excited states were characterized by time-dependent density functional theory as A-C states that had large components of Rydberg-like 3s molecular orbitals at the N-terminal and lysine ammonium groups that are conducive to hydrogen atom loss.