CHARACTERIZATION OF PROTEIN PHOSPHORYLATION OF HUMAN CHK2 PROTEIN KINASE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，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（nano-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）蛋白激酶所有Ser、Thr或Tyr残基磷酸化的理论质子化质量计算出的单、双、三带电物质的准确m/z值用于选定的离子提取和色谱分析。以激酶失活的Chk2突变体为对照，通过FT-MS和碰撞诱导解离光谱进行精确的质量测量，确定了11个Chk2自磷酸化位点。此外，从精确的质量测量中推断出在两个独特的肽中存在额外的新的Chk2磷酸化位点。所有Chk2磷酸肽的选择离子色谱都呈单峰，除了在三种情况下观察到两个紧密洗脱的物种。通过质谱分析确定这对磷酸肽为位置异构体。本研究还发现，在36个O-linked Chk2磷酸肽的MS/MS光谱中，18个由于磷酸从母体肽离子中中性损失而产生的离子并不突出。因此，精确的质量驱动分析和快速并行MS/MS采集是发现新的磷酸化位点的有用方法，这些位点与磷酸化氨基酸残基的特征损失无关。

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.