Isomeric differentiation and quantification of [alpha], [beta]-amino acid-containing tripeptides by the kinetic method: alkali metal-bound dimeric cluster ions

The kinetic method is applied to differentiate and quantify mixtures of isomeric tripeptides by generating and mass-selecting alkali metal ion-bound dimeric clusters and examining their competitive dissociations in an ion trap mass spectrometer. This methodology readily distinguishes the pairs of isomers examined here: ([alpha]-A)GG/([beta]-A)GG, G([alpha]-A)G/G([beta]-A)G, and GG([alpha]-A)/GG([beta]-A). The isomeric selectivity increases with decreasing size of the metal ion, viz. from Cs to Rb to K to Na to Li. When alanine is at the N-terminus, as in the case of ([alpha]-A)GG/([beta]-A)GG, the isomeric selectivity can exceed 103. The corresponding proton-bound dimers behave similarly to the Li clusters. Structural features that favor zwitterionic versus charge-solvated forms of the alkali metal-bound clusters are reflected in the bn and yn fragment ion abundances recorded by tandem mass spectrometry, and the propensities to form the charge-solvated or zwitterionic structures play a key role in promoting isomeric differentiation. The zwitterionic forms favor intramolecular interactions in the cluster and hence isomeric distinction. There is no discrimination in the formation of the alkali metal-bound dimers, so isomeric quantification is based entirely on dissociation kinetics. Previous kinetic method-based isomeric analyses have used the trimeric clusters and shown linear correlations between composition of the mixture of isomers and the logarithm of the branching ratio for competitive fragmentation. A similar relationship is found for the dimeric clusters examined here. As used here, the kinetic method provides a possible way for future quantitative analysis of mixtures of larger peptides such as those generated in combinatorial synthesis of peptides and peptide mimics.