In order to investigate gas-phase fragmentation reactions of phosphorylated peptide ions, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI-TOF/TOF (TOF: time-of-flight) spectra of synthetic arginine-containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C-terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI-MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI-MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C-terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI-TOF/TOF spectra of phosphopeptides displaying N-terminal fragment ions, abundant b–H3PO4 ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI-TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine-containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage-enhancing effect was observed in some lysine-containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.
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