Examining factors that influence erroneous phosphorylation site localization via competing fragmentation and rearrangement reactions during ion trap CID-MS/MS and -MS3
Examining factors that influence erroneous phosphorylation site localization via competing fragmentation and rearrangement reactions during ion trap CID-MS/MS and -MS3
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DOI：
10.1002/pmic.201200384
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发表时间：
2013-03-01
期刊：
影响因子：
3.4
通讯作者：
Reid, Gavin E.
中科院分区：
文献类型：
--
作者：
Cui, Li;Reid, Gavin E.
通过系统地改变人工合成磷肽库的肽组成、磷酸化供体和非磷酸化受体残基的身份、数目和位置以及前体离子电荷态的质子迁移率，考察了影响质子化磷肽离子CID-MS/MS和CID-MS3过程中竞争碎裂和分子内磷酸基团重排反应大小的因素，以及它们对自动搜索算法定位磷酸化位点的影响。通过HPO3和H2O的联合中性损失形成的产物离子的CID-MS3，而不是含磷酸酪氨酸的多肽直接失去H3PO4产生的产物离子的CID-MS3产生了错误的磷酸化位点分配，而含有磷酸苏氨酸和磷酸丝氨酸的多肽的正确磷酸化位点分配高度依赖于这些竞争碎片途径的相对丰度。从多个质子化的磷肽离子的CID-MS/MS中观察到丰富的磷酸基团重排产物离子，在非移动或部分移动的质子化条件下，重排增加，并作为供体和受体残基的身份和数量的函数。这些重排反应的幅度与磷酸化位点定位的置信度之间存在明显的相反趋势，重排在几个肽的错误磷酸化位点分配中起到了作用。
Factors influencing the magnitude of competing fragmentation and intramolecular phosphate group rearrangement reactions during CID-MS/MS and CID-MS3 of protonated phosphopeptide ions in ion trap mass spectrometers, and their effect on phosphorylation site localization using automated search algorithms, have been examined by systematically varying the peptide composition, the identity, number, and position of the phosphorylated donor and nonphosphorylated acceptor residues, and the proton mobility of the precursor ion charge states for a synthetic phosphopeptide library. CID-MS3 of product ions formed via combined neutral losses of HPO3 and H2O, rather than direct loss of H3PO4 from phosphotyrosine containing peptides yielded incorrect phosphorylation site assignments, while correct phosphorylation site assignments for phosphothreonine and phosphoserine containing peptides were highly dependant on the relative abundance of these competing fragmentation pathways. Abundant phosphate group rearrangement product ions were observed from CID-MS/MS of multiply protonated phosphopeptide ions, with increased rearrangement under nonmobile or partially mobile protonation conditions, and as a function of the identity and number of the donor and acceptor residues. A clear inverse trend was observed between the amplitude of these rearrangement reactions and the confidence for phosphorylation site localization, and rearrangement played a contributing role in erroneous phosphorylation site assignment for several peptides.