IMPROVEMENTS IN PROTOCOLS FOR PHOSPHOPEPTIDE MAPPING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 用质谱仪研究蛋白质磷酸化通常需要从复杂的生物样品中富集低丰度的蛋白质和多肽。一种富集法是固定化金属离子亲和层析(IMAC)。非特异性结合是IMAC浓缩过程中的一个并发症，可以通过甲醇盐酸盐处理来酯化多肽酸性基团来减少非特异性结合。对酯化过程中发生的副反应进行了调查，以记录导致这些副反应发生的条件并将其影响降至最低。已经探索了替代金属氧化物的使用，以及用钙盐沉淀磷肽/蛋白质。每个程序都通过使用标准进行了优化，然后应用于来自生物来源的样品的分析。在LTQ-Orbitrap串联MS的LC/MS分析过程中，已经开发了有效检测磷酸肽的程序。一种新的解离方法--电子转移解离(ETD)正被用于Bruker离子陷阱，并为标准和先前表征的多肽提供了出色的特定部位的结果。在P41拨款的补充支持下，它现在被添加到Orbitrap系统中。该系统正被用于分析至少部分位点尚不清楚的磷酸肽。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The study of protein phosphorylation by mass spectrometry often requires enrichment of low abundance proteins and peptides from complex biological samples. One method of enrichment is immobilized metal-ion affinity chromatography (IMAC). Non-specific binding is a complication in IMAC enrichment, and can be reduced by treatment with methanolic HCl to esterify peptide acidic groups. The side reactions that occur during esterification have been investigated to document the conditions that lead to their occurrence and minimize their impact. The use of alternative metal oxides has been explored, as well as precipitation of phosphopeptides/ proteins with calcium salts. Each procedure has been optimized by use of standards and then applied to the analysis of samples from biological sources. Procedures have been developed for efficient detection of phosphopeptides during LC/MS analysis on the LTQ-Orbitrap tandem MS. A new dissociation method, electron transfer dissociation (ETD) is being used on the Bruker ion trap and has provided excellent, site-specific results for standards and previously characterized peptides.With support from a supplement to the P41 grant, it is now being added to the Orbitrap system. This system is being used for the analysis of phosphopeptides for which at least some of the sites are as yet unknown.