A new maleimide-bound acid-cleavable solid-support reagent for profiling phosphorylation
A new maleimide-bound acid-cleavable solid-support reagent for profiling phosphorylation
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DOI：
10.1002/rcm.1869
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发表时间：
2005-01-01
影响因子：
2
通讯作者：
Bruce, JE
中科院分区：
文献类型：
--
作者：
Chowdhury, SM;Munske, GR;Bruce, JE
本文报道了一种新的磷酸肽分析化学方法。磷酸化是蛋白质翻译后修饰中最重要的一类。在这里，我们报告了一个广义的战略，采用固相捕获和质量编码的步骤，选择性地丰富复杂的混合物中的磷酸肽。该方法利用磷酸盐转化为硫醇和反应性化合物来选择性地分离磷酸化产物。磷酸肽的选择性分离是用一种简单的、新颖的、酸可裂解的、固相支持的马来酰亚胺试剂实现的。我们的化学工作集中在最小化接头大小和简化试剂生产与常见的固相肽合成步骤的结合。通过掺入乙二硫醇和丙二硫醇修饰磷酸肽来证明相对定量。我们观察到，适当的标准化是必要的，利用质量标签策略的翻译后修饰的相对定量。以磷酸肽为模型化合物，对固相捕获法的实用性进行了研究，并以β-酪蛋白、α-酪蛋白和卵清蛋白中的磷酸肽为富集对象，对固相捕获法进行了验证。固相捕获和释放的方法也证明了与未分级的整个组蛋白的蛋白质混合物，以显示该化合物在真实的生物样品中的适用性。新的化学策略最终将用于磷酸化和可能的其他翻译后修饰的高通量分析。版权所有（c）2005年约翰威利父子有限公司。
A new chemical strategy for phosphopeptide profiling is reported in this study. Phosphorylation represents one of the most important classes of posttranslational modifications of proteins. Here we report a generalized strategy that employs solid-phase capture and mass-encoding steps to selectively enrich phosphopeptides from complex mixtures. This method exploits conversion of phosphates into thiols and reactive compounds to selectively isolate products of phosphorylation. Selective isolation of phosphopeptides is achieved with a simple, novel, acid-cleavable, solid-support-bound maleimide reagent. Our chemistry efforts have focused on minimization of linker size and simplification of reagent production with incorporation of common solid-phase peptide synthesis steps. Relative quantitation was demonstrated by modifying phosphopeptides with incorporation of ethanedithiol and propanedithiol. We observed that appropriate normalization is necessary to utilize mass tag strategies for relative quantitation of posttranslational modifications. The utility of solid-phase capture was determined with model phosphopeptides, and the method was demonstrated with enriching phosphopeptides from beta-casein, alpha-casein and ovalbumin. The solid-phase capture and release methods were also demonstrated with unfractionated whole histone protein mixtures to show this compound applicability in real biological samples. The new chemical strategy will ultimately be utilized for high-throughput profiling of phosphorylation and possibly other posttranslational modifications. Copyright (c) 2005 John Wiley & Sons, Ltd.