PALMITOYLATED PROTEINS: PURIFICATION AND IDENTIFICATION

棕榈酰化蛋白质：纯化和鉴定

• 批准号: 7957766
• 负责人: NICHOLAS G DAVIS
• 金额: $ 2.09万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957766
• 关键词: Affinity; Biology; Biotin; Chemicals; Chemistry; Computer Retrieval of Information on Scientific Projects Database; Ethylmaleimide; Funding; Fungal Genome; Grant; Hydroxylamine; In Vitro; Institution; Label; Methods; Modification; Mutation; Pharmaceutical Preparations; Proteins; Proteomics; Protocols documentation; Research; Research Personnel; Resources; Source; Sulfhydryl Compounds; Technology; United States National Institutes of Health; base; link protein; palmitoylation; propionamide; protein complex; protein purification; streptavidin-agarose; tandem mass spectrometry; thioester

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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这种蛋白质组学方法从复杂的蛋白质提取物中纯化和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三个体外化学步骤取代硫酯连接的蛋白质的酰基修饰：(I)用N-乙基马来酰亚胺封闭游离的硫醇；(Ii)用羟胺切断半胱氨酸-棕榈酰硫酯的键；以及(Iii)用生物素-羟色胺(Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3‘-(2’-pyridyldithio)propionamide.标记新暴露在羟胺上的硫醇生物素化的蛋白质然后用链霉亲和素-琼脂糖亲和纯化，并通过多维蛋白质鉴定技术(MUDIT)进行鉴定，MUDIT是一种基于高通量串联质谱仪(MS/MS)的蛋白质组学技术。Mudit还提供了半定量分析，可用于评估突变或药物引起的全球棕榈酰化水平的总体变化。通常，此分析需要2-3周时间。