CHARACTERIZATION OF PROTEIN PHOSPHORYLATION OF HUMAN CHK2 PROTEIN KINASE

人 CHK2 蛋白激酶的蛋白磷酸化特征

• 批准号: 8168703
• 负责人: HELEN M PIWNICA-WORMS
• 金额: $ 0.97万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168703
• 关键词: Amino Acids; Charge; Computer Retrieval of Information on Scientific Projects Database; Data; Dissociation; Fourier transform ion cyclotron resonance; Funding; Grant; Human; Institution; Ions; Isomerism; Link; Mass Spectrum Analysis; Measurement; Methods; Parents; Peptides; Phosphopeptides; Phosphoric Acids; Phosphorylated Peptide; Phosphorylation; Phosphorylation Site; Phosphotransferases; Research; Research Personnel; Resources; Source; United States National Institutes of Health; checkpoint kinase 2; mutant; nano; novel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We employed data-dependent analysis of protein phosphorylation using rapid acquisition nano-LC-linear-quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FT-MS). 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 FT-MS and collision-induced dissociation spectra, 11 Chk2 auto-phosphorylation sites were assigned. Additionally, the presence of additional novel 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.

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