Accurate mass-driven analysis for the characterization of protein phosphorylation. Study of the human Chk2 protein kinase.
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.
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Julie B
JB
King
Julia
J
Gross
Christine M
CM
Lovly
Henry
H
Rohrs
Helen
H
Piwnica-Worms
R Reid
RR
Townsend
D000595:Amino Acid Sequence; D064447:Checkpoint Kinase 2; D017357:Cyclotrons; D006801:Humans; D007536:Isomerism; D008969:Molecular Sequence Data; D009154:Mutation; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010756:Phosphoric Acids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D010748:Phosphopeptides; D010756:Phosphoric Acids; C030242:phosphoric acid; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases