Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.
Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.
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DOI：
10.1021/pr200403c
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发表时间：
2011-11-04
影响因子：
4.4
通讯作者：
Fitzgerald, Michael C.
中科院分区：
文献类型：
--
作者：
DeArmond, Patrick D.;Xu, Ying;Strickland, Erin C.;Daniels, Kyle G.;Fitzgerald, Michael C.
鸟枪蛋白质组学方案广泛用于复杂生物样品中蛋白质的鉴定和/或定量。这里描述的是一个鸟枪蛋白质组学协议，可用于识别复杂的蛋白质混合物中的生物相关配体的蛋白质靶。该方案将定量蛋白质组学平台与共价修饰策略相结合，称为氧化速率的蛋白质稳定性（SPROX），其利用过氧化氢介导的蛋白质中甲硫氨酸侧链氧化的变性剂依赖性来评估蛋白质和蛋白质-配体复合物的热力学性质。定量蛋白质组学平台涉及使用同量异位素质量标签和含甲硫氨酸的肽增强策略。在配体结合实验中评价该方案，该实验设计用于鉴定酵母细胞裂解物中与众所周知的酶辅因子β-烟酰胺腺嘌呤二核苷酸（NAD+）结合的蛋白质。该协议还用于研究白藜芦醇的蛋白质靶点，白藜芦醇是一种生物活性配体，具有较少的蛋白质靶点。白藜芦醇的一个已知的蛋白质靶点，细胞溶质醛脱氢酶，除了其他六个潜在的新蛋白质靶点，包括四个与蛋白质翻译机制相关的蛋白质靶点，这是以前被认为是白藜芦醇的靶点。
Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme co-factor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.
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影响因子：
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