The importance of the backbone and C-terminus to mass spectrometry studies of peptides: gas-phase dissociation and acidity studies

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The work described in this dissertation shows the importance of the C-terminus and the backbone in dissociation and deprotonation of peptides. Characteristic dissociative behavior can be extremely valuable in proteomic applications and for mechanistic interpretation of mass spectra. Identification of the deprotonation site of a peptide is also important to the development of mechanisms for mass spectrometry, as dissociation is often charge-directed. Collsion-induced dissociation (CID) and electron transfer dissociation (ETD) have been used to discover distinguishing features of peptide dissociation related to the presence of an amidated C-terminus (-CONH_2) compared with the standard acid C-terminus (-COOH). Protonated peptide acids and amides are found to produce practically identical spectra, except for increased ammonia loss from the precursor for the peptide amide. ETD of multiply-protonated peptide acids and amides also produce similar spectra, although dissociation trends related to the basic amino acid residues (e.g. Arg, His, Lys) are observed for the peptide pairs. Deprotonated peptide acids and amides produced several unique product ions that differentiated the analogs. In CID experiments, abundant c_m-2^- (m = the number of amino acid residues in the peptide) formed for many of the peptide amides and c_m-3^- formed for many of the peptide acids. Supporting computational work by Michele Stover of the Dixon Group shows that the process leading to c_m-2^- from peptide amides is less endothermic than the same process for peptide acids. Gas-phase acidities (GA) have been determined for tyrosine, phenylalanine, their amino acid amides and 4-(4-hydroxyphenyl)-2-butanone using bracketing ion/molecule reactions. Two deprotonated species of tyrosine are observed, corresponding to deprotonation at the carboxylic acid -OH and the phenolic -OH. The two GAs determined for tyrosine are: GA(1) = 332.4 ± 2.2 kcal/mol and GA(2) = 333.5 ± 2.4 kcal/mol. Tyrosine amide has an experimental GA of 336.4 ± 2.7 kcal/mol, phenylalanine has a GA of 332.5 ± 2.2 kcal/mol, phenylalanine amide has a GA of 345.8 ± 3.8 kcal/mol, and 4-(4-hydroxyphenyl)-2-butanone has a GA of 339.6 ± 3.0 kcal/mol. The GAs of six tripeptides (with alkyl or H- side chains) have been determined. All of the experimental GAs fall within a 1.2 kcal/mol range, which is consistent with the C-terminus being the most acidic site on the peptides. The GAs of three methyl esters have been determined, demonstrating the ability of peptides to deprotonate on the backbone. The peptide methyl ester GAs are all very similar and fall within a 1.4 kcal/mol range. Computational results indicate that these methyl esters are deprotonating at the central amide NH. Three other methyl esters could not be deprotonated by ESI, because of conformation and steric hindrance to the deprotonation site.

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Analytical chemistry