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: 7953916
• 负责人: HELEN M PIWNICA-WORMS
• 金额: $ 0.58万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7953916
• 关键词: Amino Acids; Charge; Computer Retrieval of Information on Scientific Projects Database; Cyclic AMP-Dependent Protein Kinases; 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; biomedical resource; 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我们使用快速采集纳米-LC线性 - Quadrupole离子陷阱傅里叶转化离子离子共振质谱法（纳米-LC-FT-MS）对蛋白质磷酸化进行了数据依赖性分析。从所有Ser，Thr，THR或Tyr的磷酸化的理论质子化质子块（CHK2）蛋白激酶的磷酸化理论质子化质量计算出的单一，双重和三重电荷物种的准确M/Z值被用于选定的离子提取和色谱分析。 使用激酶不活跃的CHK2突变体作为对照，从FT-MS和碰撞诱导的解离光谱中进行了准确的质量测量，分配了11个CHK2自磷酸化位点。 此外，从准确的质量测量中推导了两个独特的肽中其他新型CHK2磷酸化位点的存在。 所有CHK2磷酸肽的选定离子色谱图给出了单个峰，但在三种情况下观察到了两种紧密洗脱物种。 通过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O-Chinked CHK2磷酸肽的36 ms/ms光谱中的18个中，由于来自母肽离子的中性磷酸丧失而引起的离子并不突出。 因此，准确的质量驱动分析和快速平行的MS/MS采集是发现新的磷酸化位点的有用方法，该方法与磷酸化氨基酸残基的特征损失无关。