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Examining factors that influence erroneous phosphorylation site localization via competing fragmentation and rearrangement reactions during ion trap CID ‐ MS / MS and ‐ MS 3
Author(s) -
Cui Li,
Reid Gavin E.
Publication year - 2013
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201200384
Subject(s) - phosphopeptide , chemistry , fragmentation (computing) , phosphorylation , protonation , ion , mass spectrometry , chromatography , biochemistry , organic chemistry , biology , ecology
Factors influencing the magnitude of competing fragmentation and intramolecular phosphate group rearrangement reactions during CID ‐ MS / MS and CID ‐ MS 3 of protonated phosphopeptide ions in ion trap mass spectrometers, and their effect on phosphorylation site localization using automated search algorithms, have been examined by systematically varying the peptide composition, the identity, number, and position of the phosphorylated “donor” and nonphosphorylated “acceptor” residues, and the proton mobility of the precursor ion charge states for a synthetic phosphopeptide library. CID ‐ MS 3 of product ions formed via combined neutral losses of HPO 3 and H 2 O , rather than direct loss of H 3 PO 4 from phosphotyrosine containing peptides yielded incorrect phosphorylation site assignments, while correct phosphorylation site assignments for phosphothreonine and phosphoserine containing peptides were highly dependant on the relative abundance of these competing fragmentation pathways. Abundant phosphate group rearrangement product ions were observed from CID ‐ MS / MS of multiply protonated phosphopeptide ions, with increased rearrangement under nonmobile or partially mobile protonation conditions, and as a function of the identity and number of the donor and acceptor residues. A clear inverse trend was observed between the amplitude of these rearrangement reactions and the confidence for phosphorylation site localization, and rearrangement played a contributing role in erroneous phosphorylation site assignment for several peptides.