Investigations described in this dissertation found that cleavage at Xxx-Pro bonds depends upon the identity of the Xxx residue, and this finding is immediately applicable to improved peptide sequencing algorithms because enhanced cleavage was evident for Pro-containing peptides when Xxx is His, Asp, Val, Ile, or Leu, and reduced cleavage was evident when Xxx is Gly or Pro. A greater understanding of the mechanism of cleavage at Pro residues was gained in these studies. A non-Pro N-alkylated residue in a peptide directs cleavage C-terminal to the N-alkylated residue; this is dependent upon the conformation of the peptide as cleavage is directed N-terminal to the residue when the D-stereoisomer of the N-alkylated residue is substituted into the peptide. Cleavage rarely occurs C-terminal to Pro residues in peptides, presumably due to steric limitations that are imposed by the 5-membered ring and that hinder formation of necessary intermediates. Dramatically different gas-phase H/D exchange results observed for L-Pro-containing peptides after substitution with the D-stereoisomer of Pro in addition to evidence of Pro-cleavage dependence upon the residue preceding Pro and conformational dependence of cleavage of other N-alkylated residues leads us to conclude that the conformational turn in a peptide backbone and the steric limitations imposed by the Pro ring are the primary factors affecting cleavage at Pro. Fragmentation patterns of peptides containing Pro and other N-alkylated residues support a mechanism of bond cleavage that begins with protonation at a carbonyl oxygen rather than an amide nitrogen. Transfer of the ionizing proton (or its equivalent) to the C-terminal fragment forms "b" ions which may be followed by the transfer of the hydrogen bound to the amide nitrogen N-terminal to the cleavage site for "y" ion formation. The types of experiments designed to investigate Pro-containing peptides also provided new results that answered questions about gas-phase structure of other peptides. Gas-phase H/D exchange of Arg-containing oligoalanines and their derivatives suggests that salt bridge structures may form for these 5-residue peptides. MS3 and gas-phase H/D exchange results of fragment ions from Asp-containing peptides suggest different "b" ion structures formed at Asp-Xxx and non-Asp cleavage positions
