Abstract
MS/MS fragmentation of peptides is dominated by overlapping b and y ion series. However, alternative fragmentation possibilities exist, including neutral loss. A database was generated containing 8400 MS/MS spectra of tryptic peptides assigned with high probability to an amino acid sequence (true positives) and a set of certified false (true negative) assignments for analysis of the amino terminus. A similar database was created for analysis of neutral loss at the carboxy termini using a data set of chymotryptic peptides. The analysis demonstrated that the presence of an internal basic residue, limiting proton mobility, has a profound effect on neutral loss. Peptides with fully mobile protons demonstrated minimal neutral loss, with the exception of amide bonds with proline on the carboxy terminal side, which created an intense neutral loss peak. In contrast, peptides with partial proton mobility contained many amino acids on either side of the amide bond associated with a strong neutral loss peak. Most notable among these was proline on the carboxy terminal side of an amide bond and aspartic acid on the amino terminal side of a bond. All results were found to be consistent for doubly and triply charged peptides and after adjustment for pairings across the amide bonds with particularly labile residues. The carboxy terminal of chymotryptic peptides also demonstrated significant neutral loss events associated with numerous amino acid residues. Clarification of the rules that govern neutral loss, when incorporated into analysis software, will improve our ability to correctly assign spectra to peptide sequences.