Phosphorylated serine and threonine residues promote site-specific fragmentation of singly charged, arginine-containing peptide ions.
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-H(3)PO(4) 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.
Gehrig
Peter Max
PM
Functional Genomics Center Zurich, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. peter.gehrig@fgcz.uzh.ch
Roschitzki
Bernd
B
Rutishauser
Dorothea
D
Reiland
Sonja
S
Schlapbach
Ralph
R
D001120:Arginine / Q000737:chemistry*; D007477:Ions / Q000737:chemistry*; D012275:Oryza / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013912:Threonine / Q000737:chemistry*
D007477:Ions; D010748:Phosphopeptides; D010940:Plant Proteins; D013912:Threonine; D012694:Serine; D001120:Arginine