A two-step neutral loss from tryptic peptides containing Thr-Thr or Thr-Ser at their N-terminus is studied. This process also requires a mobile proton (i.e., the number of charges on the peptide is greater than the number of basic amino acids) and leads to a net neutral loss of 62 Da. To elucidate this fragmentation a series of synthetic peptides containing threonine and serine were synthesized and their MS/MS spectra measured in ion trap and triple quadrupole instruments. Peptides composed of TTLnK (n = 2 to 8) all show a significant loss of 62 Da when doubly protonated, but little such loss when singly protonated. Examination of the MS/MS spectra at different collision energies in a triple quadrupole mass spectrometer reveal that this loss takes place in two distinct steps: an initial water loss, followed by a loss of a 44 Da moiety at higher collision energies (20 - 30 % higher). Corresponding losses in b/a ions show losses near the N-terminus and higher accuracy mass measurements indicate the loss to be C2H4O rather than CO2. Further measurements show that doubly protonated TSLnK peptides undergo similar processes but STLnK and SSLnK do not. Therefore, it is proposed that the 44 Da loss is a loss of C2H4O from the N-terminal threonine. Additional measurements on the loss of water from protonated peptides containing only one threonine or serine show a strong and unexpected dependence of the rate of water loss on the position of T or S within the peptide sequence. The most pronounced water loss is found in doubly protonated tryptic peptides containing T or S at the second or third position from the N-terminus, i.e. adjacent to the peptide bond that is most likely to cleave.
Citation: International Journal of Mass Spectrometry
Pub Type: Journals
fragmentation, mass spectrometry, neutral loss, peptide ions, peptides