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: 7355307
• 负责人: HELEN M PIWNICA-WORMS
• 金额: $ 0.34万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355307
• 关键词: CHARACTERIZATION; PROTEIN; PHOSPHORYLATION; HUMAN; CHK2

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.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们利用快速获取的纳米LC-线性四极离子陷阱傅立叶变换离子回旋共振质谱仪(Nano-LC-FT-MS)对蛋白质磷酸化进行数据相关分析。根据人Chk2蛋白激酶所有Ser、Thr或Tyr残基的理论质子化质量计算出单电荷、双电荷和三电荷物种的精确m/z值，用于选择性离子萃取和色谱分析。以一个激酶失活的Chk2突变体为对照，通过FT-MS的准确质量测量和碰撞诱导解离光谱，指定了11个Chk2自动磷酸化位点。此外，从准确的质量测量中推断出在两个独特的多肽中存在额外的新的Chk2磷酸化位点。除3例观察到两个紧密洗脱物种外，所有Chk2磷酸肽的选择性离子色谱图均为单峰。通过MS/MS分析，确定这对磷酸肽为位置异构体。在本研究中还发现，在36个O-连接的Chk2磷酸肽的MS/MS图谱中，有18个并不突出，这是由于磷酸从母体失去中性离子而引起的。因此，准确的质量驱动分析和快速并行的MS/MS获取对于发现新的与磷酸化氨基酸残基的特征损失无关的磷酸化位点是一种有用的方法。