Evaluation of Gas-Phase Rearrangement and Competing Fragmentation Reactions on Protein Phosphorylation Site Assignment Using Collision Induced Dissociation-MS/MS and MS3
Evaluation of Gas-Phase Rearrangement and Competing Fragmentation Reactions on Protein Phosphorylation Site Assignment Using Collision Induced Dissociation-MS/MS and MS3
复制标题
DOI：
10.1021/ac801768s
复制
发表时间：
2008-12-15
影响因子：
7.4
通讯作者：
Reid, Gavin E.
中科院分区：
文献类型：
--
作者：
Palumbo, Amanda M.;Reid, Gavin E.
基于离子阱串联质谱的常规磷酸肽鉴定技术的最新进展无疑促进了磷蛋白质组综合分析策略的发展。然而，当磷酸化肽序列中存在多个潜在的磷酸化位点时，磷酸化位点的明确表征仍然是一个重大挑战。本文利用碰撞诱导解离（CID）和多级串联质谱（MS/MS和MS3）在线性四极离子阱中研究了一系列含有多个潜在磷酸化位点的33种合成磷酸丝氨酸、-苏氨酸和-酪氨酸肽的气相裂解反应。在这项研究中，15个肽（45%）产生产物离子，这些产物离子是在CID-MS/MS上磷酸基从磷酸化残基转移到未修饰的含羟基氨基酸残基后形成的。这种重排的倾向被发现高度依赖于前体离子电荷状态和肽的氨基酸组成（即质子迁移率），并且主要观察到在“非移动”或“部分移动”质子化条件下的肽。这些重排反应的观察和/或产物离子的缺乏为正确的磷酸化位点提供了明确的证据，限制了明确分配正确磷酸化位点的能力，只有33个肽中的12个（36%）。此外，观察到这些前体离子对98 Da中性损失的竞争性断裂反应（即H3PO4的损失与HPO3和H2O的联合损失）表明[M + nH - 98]（n+）离子的CID-MS3可能不能用于明确的磷酸化位点定位。
The development of strategies directed toward comprehensive analysis of the phosphoproteome have undoubtedly been facilitated by recent advances in the application of ion trap tandem mass spectrometry-based techniques for routine phosphopeptide identification. However, when multiple potential sites of phosphorylation exist within a phosphorylated peptide sequence, unambiguous characterization of the site of phosphorylation remains a significant challenge. Here, the gas-phase fragmentation reactions of a series of 33 synthetic phospho-serine, -threonine, and -tyrosine peptides containing multiple potential phosphorylation sites have been examined using collision induced dissociation (CID) and multistage tandem mass spectrometry (MS/MS and MS3) in a linear quadrupole ion trap. From this study, 15 of the peptides (45%) gave rise to product ions that were formed following initial transfer of a phosphate group from the phosphorylated residue to an unmodified hydroxyl-containing amino acid residue upon CID-MS/MS. The propensity for this rearrangement was found to be highly dependent on the precursor ion charge state and amino acid composition (i.e, proton mobility) of the peptide and was observed predominantly for peptides under "nonmobile" or "partially mobile" protonation conditions. The observation of these rearrangement reactions and/or the lack of product ions that provided definitive evidence for the correct site of phosphorylation, limited the ability to unambiguously assign the correct site of phosphorylation to only 12 of the 33 peptides (36%). Furthermore, the observation of competing fragmentation reactions for the neutral loss of 98 Da from these precursor ions (i.e., the loss of H3PO4 versus the combined losses of HPO3 and H2O) indicates that CID-MS3 of [M + nH - 98](n+) ions may not be used for unambiguous phosphorylation site localization.