Premium
A combination of neutral loss and targeted product ion scanning with two enzymatic digestions facilitates the comprehensive mapping of phosphorylation sites
Author(s) -
CasadoVela Juan,
Ruiz Edgar J.,
Nebreda Angel R.,
Casal J. Ignacio
Publication year - 2007
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.200700026
Subject(s) - phosphorylation , cyclin dependent kinase 1 , phosphopeptide , chemistry , cyclin dependent kinase , phosphatase , biochemistry , kinase , trypsin , protein phosphorylation , enzyme , biology , protein kinase a , cell cycle , cell
We propose here a new strategy for the exhaustive mapping of phosphorylation sites in the Xenopus laevis Cdc25 phosphatase, which regulates cell cycle progression in eukaryotic cells. Two different MS analyses in a linear IT were used to identify the phosphorylated residues. First, a data‐dependent neutral loss (DDNL) analysis triggered the fragmentation of peptides that show enhanced neutral loss of phosphoric acid. Second, a targeted product ion scanning (TPIS) mass analysis was carried out in which MS 2 events are triggered for specific m / z values. Full coverage of the protein sequence was obtained by combining the two analyses with two enzymatic digestions, trypsin and chymotrypsin, yielding a comprehensive map of the phosphorylation sites. Previous reports have shown Cdc25C to be phosphorylated by Cdc2–cyclin B at four residues (Thr 48 , Thr 67 , Thr 138 and Ser 205 ). By using this combination of scan modes, we have identified four additional phosphorylation sites (Thr 86 , Ser 99 , Thr 112 and Ser 163 ) in a recombinant Cdc25C protein containing 198 residues of the NH 2 ‐terminal noncatalytic domain. The sensitivity of this combined approach makes it extremely useful for the comprehensive characterization of phosphorylation sites, virtually permitting complete coverage of the protein sequence with peptides within the mass detection range of the linear IT.