Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis
Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis
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DOI：
10.1002/pmic.200500073
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发表时间：
2006-02-01
期刊：
影响因子：
3.4
通讯作者：
James, P
中科院分区：
文献类型：
--
作者：
Arrigoni, G;Resjö, S;James, P
蛋白质磷酸化是调节细胞中蛋白质功能的最重要和最常见的方式之一。然而，磷酸肽很难分析，在标准MALDI条件下电离性差。为了解决磷酸肽分析的低灵敏度和低特异性的问题，已经开发了几种方法。在这里，我们展示了一种使用简单的一步β -消除/Michael加成反应衍生化磷丝氨酸和磷苏氨酸的方法。带负电的磷酸基团被带正电的s -乙基吡啶基取代，极大地改善了修饰肽的电离性，特别是在MALDI MS中，提高了分析的灵敏度。该修饰允许在温和的碰撞激活条件下在m/ z106处形成独特的碎片离子，可用于母体（前体）离子扫描，以提高分析的灵敏度和选择性。该方法的优化描述了一个标准模型肽和蛋白质，然后应用于磷酸化分析在两个生物来源的蛋白质从不同的实验系统纯化。
Protein phosphorylation is one of the most important and common ways of regulating protein function in cells. However, phosphopeptides are difficult to analyse, ionising poorly under standard MALDI conditions. Several methods have been developed to deal with the low sensitivity and specificity of phosphopeptide analysis. Here, we show an approach using a simple one-step beta-elimination/Michael addition reaction for the derivatization of phosphoserine and phosphothreonine. The substitution of the negatively charged phosphate group by a positively charged S-ethylpyridyl group greatly improves the ionisation of the modified peptides, especially in MALDI MS, increasing the sensitivity of the analysis. The modification allows the formation of a unique fragment ion at m/z 106 under mild collisional activation conditions, which can be used for parent (precursor) ion scanning in order to improve both the sensitivity and the selectivity of the analysis. The optimisation of the approach is described for a standard model peptide and protein and then applied to phosphorylation analysis in two biologically derived proteins purified from different experimental systems.