Analysis of phosphorylated peptides by double pseudoneutral loss extraction coupled with derivatization using N-(4-bromobenzoyl)aminoethanethiol.
Analysis of phosphorylated peptides by double pseudoneutral loss extraction coupled with derivatization using N-(4-bromobenzoyl)aminoethanethiol.
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DOI：
10.1021/ac9017988
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发表时间：
2009-10
影响因子：
7.4
通讯作者：
N. Mano;Sayaka Aoki;Takuma Yamazaki;Yoko Nagaya;Masaru Mori;K. Abe;M. Shimada;Hiroaki Yamaguchi;T. Goto;J. Goto
中科院分区：
文献类型：
--
作者：
N. Mano;Sayaka Aoki;Takuma Yamazaki;Yoko Nagaya;Masaru Mori;K. Abe;M. Shimada;Hiroaki Yamaguchi;T. Goto;J. Goto
翻译后磷酸化的分析是理解许多生理事件机制的关键步骤。文献中报道了许多旨在通过质谱检测和定量磷酸化蛋白质的分析方法的开发方法。在本文中，我们报告了一种鉴定磷蛋白中磷酸化丝氨酸和苏氨酸残基的新策略。该方法包括磷蛋白的选择性衍生化以及 nanoLC/ESI-MS/MS 分析后的双假中性损失提取。首先，我们设计并合成了一种新的衍生试剂N-(4-溴苯甲酰基)氨基乙硫醇，它可以选择性地与磷酸化丝氨酸或苏氨酸残基中磷酰基的β-消除产生的α,β-不饱和酮反应。衍生肽的质谱显示具有特征同位素模式的产物离子。衍生化后，含有磷酸丝氨酸或磷酸苏氨酸的肽的碎片离子具有双峰，强度比约为1:1，相差两个质量单位，而不含磷酸丝氨酸或磷酸苏氨酸的肽的产物离子具有正常的同位素模式。因此，产物离子质谱中衍生残基的中性损失包括由(79)Br和(81)Br引起的两个差异损失。具有双假中性损失的产物扫描质谱的提取对于鉴定由磷酸化肽产生的衍生肽非常有效。新策略代表了分析磷酸化蛋白质中磷酸化丝氨酸和苏氨酸残基的有用工具。
The analysis of post-translational phosphorylation is a crucial step in understanding the mechanisms of many physiological events. Numerous approaches for the development of analytical methods aimed at the detection and quantification of phosphorylated proteins by mass spectrometry have been reported in the literature. In this paper, we report a new strategy for the identification of phosphorylated serine and threonine residues in phosphoproteins. This method consists of selective derivatization of phosphoproteins coupled with double pseudoneutral loss extraction after nanoLC/ESI-MS/MS analysis. First, we designed and synthesized a new derivatization reagent, N-(4-bromobenzoyl)aminoethanethiol, which can selectively react with alpha,beta-unsaturated ketones produced by beta-elimination of a phosphoryl group from phosphorylated serine or threonine residues. The mass spectrum of the derivatized peptide shows a product ion with a characteristic isotopic pattern. After derivatization, fragment ions of peptides with phosphoserine or phosphothreonine have twin peaks with an intensity ratio of approximately 1:1 and a difference of two mass units, while product ions from peptides without phosphoserine or phosphothreonine have normal isotopic patterns. Therefore, the neutral loss of the derivatized residue in the product ion mass spectrum includes two difference losses caused by (79)Br and (81)Br. The extraction of the product scan mass spectrum with double pseudoneutral losses is very effective for identification of the derivatized peptides produced from phosphorylated peptides. The new strategy represents a useful tool for the analysis of phosphorylated serine and threonine residues in phosphorylated proteins.