Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent.
Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent.
复制标题
使用磺酸盐掺入试剂通过双喷雾离子/离子反应调节磷酸肽断裂。
DOI：
10.1021/acs.analchem.6b01901
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发表时间：
2016
影响因子：
7.4
通讯作者：
Brodbelt,JenniferS
中科院分区：
文献类型：
--
作者：
Cotham,VictoriaC;McGee,WilliamM;Brodbelt,JenniferS
磷酰基的不稳定性给传统的基于质谱的自下而上的蛋白质组学方法表征蛋白质磷酸化带来了长期的挑战。碰撞诱导解离（CID）导致肽的磷酸酯键的优先裂解，特别是在低质子迁移率的条件下，并导致抑制序列信息片段化，其通常禁止磷酸位点测定。在本研究中，通过离子/离子介导的肽衍生化与4-甲酰基-1，3-苯二磺酸（FBDSA）阴离子使用双喷雾反应器的磷酸肽的裂解模式进行了改进。这种方法利用FBDSA的磺酸盐部分和碱性位点之间的强静电相互作用，以促进气相生物缀合，并减少电荷螯合和增加CID时磷酸盐保留序列离子的产率。此外，未修饰的磷酸肽和那些修改在线FBDSA或在溶液中通过氨甲酰化和4-磺苯基异硫氰酸酯（SPITC）之间的比较CID碎片分析提供了证据磺酸盐干扰的电荷导向的机制，导致优先消除磷酸盐。我们的研究结果表明，在磷酸盐中性损失涉及的电荷导向的相邻基团参与反应的突出性，并在双喷雾反应器设置中的离子/离子反应的实施提供了一种手段，通过竞争磺酸盐基团和碱性残基的侧链之间的氢键相互作用来破坏相互作用。
The labile nature of phosphoryl groups has presented a long-standing challenge for the characterization of protein phosphorylation via conventional mass spectrometry-based bottom-up proteomics methods. Collision-induced dissociation (CID) causes preferential cleavage of the phospho-ester bond of peptides, particularly under conditions of low proton mobility, and results in the suppression of sequence-informative fragmentation that often prohibits phosphosite determination. In the present study, the fragmentation patterns of phosphopeptides are improved through ion/ion-mediated peptide derivatization with 4-formyl-1,3-benezenedisulfonic acid (FBDSA) anions using a dual spray reactor. This approach exploits the strong electrostatic interactions between the sulfonate moieties of FBDSA and basic sites to facilitate gas-phase bioconjugation and to reduce charge sequestration and increase the yield of phosphate-retaining sequence ions upon CID. Moreover, comparative CID fragmentation analysis between unmodified phosphopeptides and those modified online with FBDSA or in solution via carbamylation and 4-sulfophenyl isothiocyanate (SPITC) provided evidence for sulfonate interference with charge-directed mechanisms that result in preferential phosphate elimination. Our results indicate the prominence of charge-directed neighboring group participation reactions involved in phosphate neutral loss, and the implementation of ion/ion reactions in a dual spray reactor setup provides a means to disrupt the interactions by competing hydrogen-bonding interactions between sulfonate groups and the side chains of basic residues.