Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization
Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization
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DOI：
10.1007/s13361-011-0190-0
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发表时间：
2012-04-01
影响因子：
3.2
通讯作者：
Reid, Gavin E.
中科院分区：
文献类型：
--
作者：
Lu, Yali;Zhou, Xiao;Reid, Gavin E.
胺特异性肽衍生化策略涉及使用新型同量异位稳定同位素编码的“固定电荷”锍离子试剂，并结合采用毛细管 HPLC、ESI-MS 和自动数据依赖离子阱 CID-MS/MS、-MS3 和/或 ETD-MS/MS 的分析策略，已开发用于改进蛋白质磷酸化的定量分析，并用于鉴定和表征其修饰位点。与非衍生化磷酸肽相比，用 S,S'-二甲基硫代丁酰基羟基琥珀酰亚胺酯碘化物 (DMBNHS) 对 50 种合成磷酸肽进行衍生化，然后使用毛细管 HPLC-ESI-MS 进行分析，电离效率平均提高 2.5 倍，并且较高电荷态前体离子的存在和/或丰度显着增加。值得注意的是，44% 的磷酸肽（50 种中的 22 种）处于未衍生化状态，产生了最大电荷态对应于 +2 的前体离子，而在 DMBNHS 衍生化后，只有 8%（50 种中的 4 种）保持在该最大电荷态。通过测量诊断产物离子的丰度来实现定量分析，该诊断产物离子对应于“轻”(S(CH3)(2))和“重”(S(CD3)(2))二甲硫醚的中性损失，这些二甲基硫醚是在DMBNHS衍生化磷酸肽的同量异位稳定同位素标记形式的CID-MS/MS上专门形成的。在这些条件下，磷酸基团保持完整。由于较高电荷态前体离子的形成，通过自动化数据依赖性 CID-MS3 或 ETD-MS/MS 分析，可以获取更多数量的肽，以提供增强的磷酸肽序列鉴定和磷酸化位点表征。重要的是，在引入锍离子固定电荷后，使用 ETD-MS/MS 观察到序列覆盖率得到改善，但对 ETD 裂解效率没有不利影响。
An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded 'fixed charge' sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S'-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at thismaximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of 'light' (S(CH3)(2)) and 'heavy' (S(CD3)(2)) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency.