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: 7721480
• 负责人: HELEN M PIWNICA-WORMS
• 金额: $ 0.39万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721480
• 关键词: Amino Acids; Charge; Computer Retrieval of Information on Scientific Projects Database; Cyclotrons; Data; Dissociation; Fourier Transform; 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们使用快速采集纳米LC线性四极离子阱傅里叶变换离子回旋共振质谱（纳米LC-FT-MS）对蛋白质磷酸化进行数据依赖分析。根据人类检查点 2 (Chk2) 蛋白激酶的所有 Ser、Thr 或 Tyr 残基处磷酸化的肽的理论质子化质量计算出单电荷、双电荷和三电荷种类的准确 m/z 值，用于选择离子提取和色谱分析分析。 使用激酶失活的 Chk2 突变体作为对照，通过 FT-MS 和碰撞诱导解离谱进行精确质量测量，分配了 11 个 Chk2 自磷酸化位点。 此外，通过精确的质量测量推断出两种独特肽中存在额外的新型 Chk2 磷酸化位点。 所有 Chk2 磷酸肽的选定离子色谱图均显示单峰，但观察到两种紧密洗脱的三种情况除外。 MS/MS 分析确定这些磷酸肽对是位置异构体。 在这项研究中，还发现由于母体肽离子中磷酸中性损失而产生的离子在 O-连接 Chk2 磷酸肽的 36 个 MS/MS 谱图中的 18 个中并不突出。 因此，精确的质量驱动分析和快速并行 MS/MS 采集是发现新磷酸化位点的有用方法，该位点与磷酸化氨基酸残基的特征丢失无关。