%0 Journal Article
%A King, Julie B.
%A Gross, Julia
%A M. Lovly, Christine
%A Rohrs, Henry
%A Piwnica-Worms, Helen
%A Townsend, R. Reid
%D 2006
%T Accurate Mass-Driven Analysis for the
Characterization of Protein Phosphorylation. Study
of the Human Chk2 Protein Kinase
%U https://acs.figshare.com/articles/journal_contribution/Accurate_Mass_Driven_Analysis_for_the_Characterization_of_Protein_Phosphorylation_Study_of_the_Human_Chk2_Protein_Kinase/3229798
%R 10.1021/ac051520l.s001
%2 https://ndownloader.figshare.com/files/5063866
%K FTMS
%K Human Chk 2 Protein Kinase
%K 11 novel Chk 2 autophosphorylation sites
%K Chk 2 phosphopeptides
%K mass measurements
%K ion cyclotron resonance mass spectrometry
%K Chk 2 phosphorylation sites
%K Selected ion chromatograms
%K MS
%K parent peptide ion
%K analysis
%X We describe the data-dependent analysis of protein phosphorylation using rapid-acquisition nano-LC−linear quadrupole ion trap Fourier transform ion cyclotron resonance
mass spectrometry (nano-LC−FTMS). The accurate m/z
values of singly, doubly, and triply charged species
calculated from the theoretical protonated masses of
peptides phosphorylated at all Ser, Thr, or Tyr residues
of the human checkpoint 2 (Chk2) protein kinase were
used for selected ion extraction and chromatographic
analysis. Using a kinase-inactive Chk2 mutant as a
control, accurate mass measurements from FTMS and
collision-induced dissociation spectra, 11 novel Chk2
autophosphorylation sites were assigned. Additionally, the
presence of additional Chk2 phosphorylation sites in two
unique peptides was deduced from accurate mass measurements. Selected ion chromatograms of all Chk2
phosphopeptides gave single peaks except in three cases
in which two closely eluting species were observed. These
pairs of phosphopeptides were determined to be positional isomers from MS/MS analysis. In this study, it was
also found that ions due to the neutral loss of phosphoric
acid from the parent peptide ion were not prominent in
18 of 36 MS/MS spectra of O-linked Chk2 phosphopeptides. Thus, accurate mass-driven analysis and rapid
parallel MS/MS acquisition is a useful method for the
discovery of new phosphorylation sites that is independent
of the signature losses from phosphorylated amino acid
residues.
%I ACS Publications