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Highlights: [•] Fragmentation of phosphoramidate-containing peptides by CID in a QIT was assessed. [•] pHis homodimer formation facilitates intermolecular phosphate transfer during CID. [•] Dimer formation and phosphate transfer is dependent on a C-terminal basic Lys. [Copyright &y& Elsevier]

InfraRed Multiple Photon Dissociation (IRMPD) spectroscopy was used to assay the structural features of the fragment ions resulting from the elimination of H3PO4 in the Collision-Induced Dissociation (CID) of protonated serine. The results are interpreted with the aid of density functional theory calculations. Experiment and theory point to an aziridine-ring structure, implying participation of the vicinal amino group in the formation of this species. This finding constitutes a benchmark for investigating the same process in the CID of phosphorylated peptides. [ABSTRACT FROM AUTHOR]

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (HPO), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the HPO elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of HPO upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]