King Julie B, Gross Julia, Lovly Christine M, Rohrs Henry, Piwnica-Worms Helen, Townsend R Reid
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Anal Chem. 2006 Apr 1;78(7):2171-81. doi: 10.1021/ac051520l.
We describe the data-dependent analysis of protein phosphorylation using rapid-acquisition nano-LC-linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FTMS). 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 FTMS and collision-induced dissociation spectra, 11 novel Chk2 autophosphorylation sites were assigned. Additionally, the presence of additional 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.
我们描述了使用快速采集的纳升级液相色谱-线性四极杆离子阱傅里叶变换离子回旋共振质谱仪（纳升级液相色谱-傅里叶变换质谱仪，nano-LC-FTMS）对蛋白质磷酸化进行数据依赖分析的方法。从人检验点2（Chk2）蛋白激酶所有丝氨酸（Ser）、苏氨酸（Thr）或酪氨酸（Tyr）残基磷酸化肽段的理论质子化质量计算得到的单电荷、双电荷和三电荷物种的精确质荷比（m/z）值，用于选定离子提取和色谱分析。以激酶失活的Chk2突变体作为对照，通过傅里叶变换质谱仪的精确质量测量和碰撞诱导解离光谱，确定了11个新的Chk2自磷酸化位点。此外，从精确质量测量中推断出两个独特肽段中存在额外的Chk2磷酸化位点。除了在3个案例中观察到两个紧密洗脱的物种外，所有Chk2磷酸肽的选定离子色谱图均给出单峰。通过串联质谱（MS/MS）分析确定这些成对的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光谱中，有18个光谱中母肽离子磷酸的中性丢失产生的离子不明显。因此，精确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一种有用方法，该方法独立于磷酸化氨基酸残基的特征性丢失。
