PALMITOYLATED PROTEINS: PURIFICATION AND IDENTIFICATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该蛋白质组方案纯化并鉴定棕榈酰化蛋白质（即，S-酰化蛋白质）。该方法依赖于酰基-生物素基交换化学，其中生物素部分通过一系列三个体外化学步骤取代硫酯连接的蛋白质酰基修饰：（i）用N-乙基马来酰亚胺阻断游离巯基;（ii）用羟胺裂解Cys-棕榈酰硫酯键;和（iii）用生物素-HPDP（生物素-HPDP-N-[6-（生物素酰胺基）己基]-3 ′-（2 ′-吡啶基二硫代）丙酰胺）标记新被羟胺暴露的硫醇。然后使用链霉亲和素-琼脂糖亲和纯化生物素化的蛋白质，并通过多维蛋白质鉴定技术（MuDPIT）进行鉴定，这是一种基于高通量串联质谱（MS/MS）的蛋白质组学技术。MuDPIT还提供了一种半定量分析，可用于评估突变或药物诱导的整体棕榈酰化谱的总体变化。通常，这种分析需要2-3周。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proteomic protocol purifies and identifies palmitoylated proteins (i.e., S-acylated proteins) from complex protein extracts. The method relies on an acyl-biotinyl exchange chemistry in which biotin moieties are substituted for the thioester-linked protein acyl-modifications through a sequence of three in vitro chemical steps: (i) blockade of free thiols with N-ethylmaleimide; (ii) cleavage of the Cys-palmitoyl thioester linkages with hydroxylamine; and (iii) labeling of thiols, newly exposed by the hydroxylamine, with biotin-HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide. The biotinylated proteins are then affinity-purified using streptavidin-agarose and identified by multi-dimensional protein identification technology (MuDPIT), a high-throughput, tandem mass spectrometry (MS/MS)-based proteomic technology. MuDPIT also affords a semi-quantitative analysis that may be used to assess the gross changes induced to the global palmitoylation profile by mutation or drugs. Typically, 2-3 weeks are required for this analysis.