CHARACTERIZATION OF PROTEIN PHOSPHORYLATION OF HUMAN CHK2 PROTEIN KINASE

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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。 我们采用快速采集纳米LC-线性四极杆离子阱傅立叶变换离子回旋共振质谱（nano-LC-FT-MS）的蛋白质磷酸化的数据依赖性分析。单、双和三重电荷物质的精确m/z值，根据人检查点2（Chk 2）蛋白激酶的所有Ser、Thr或Tyr残基磷酸化肽的理论质子化质量计算，用于选择离子提取和色谱分析。 使用激酶失活的Chk 2突变体作为对照，从FT-MS和碰撞诱导解离光谱的精确质量测量，11个Chk 2自身磷酸化位点被分配。 此外，在两个独特的肽中存在额外的新的Chk 2磷酸化位点是从精确的质量测量推断的。 所有Chk 2磷酸肽的选择离子色谱图给出了单峰，除了在三种情况下，其中两个密切的洗脱物种观察。 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，由于磷酸从母体肽离子的中性损失的离子在O-连接的Chk 2磷酸肽的36个MS/MS谱中的18个中不突出。 因此，准确的质量驱动的分析和快速并行MS/MS采集是一种有用的方法，用于发现新的磷酸化位点，这是独立于从磷酸化的氨基酸残基的签名损失。

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.