An Azido-Biotin Reagent for Use in the Isolation of Protein Adducts of Lipid-derived Electrophiles by Streptavidin Catch and Photorelease
An Azido-Biotin Reagent for Use in the Isolation of Protein Adducts of Lipid-derived Electrophiles by Streptavidin Catch and Photorelease
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DOI：
10.1074/mcp.m900121-mcp200
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发表时间：
2009-09-01
影响因子：
7
通讯作者：
Porter, Ned A.
中科院分区：
文献类型：
--
作者：
Kim, Hye-Young H.;Tallman, Keri A.;Porter, Ned A.
HNE（4-羟基壬烯醛）是脂质过氧化的副产物，与蛋白质上的亲核中心发生反应。 HNE 的末端炔基类似物（炔基 HNE，aHNE）可作为 HNE 本身的替代物，这两种化合物通过类似的化学反应与蛋白胺和硫醇官能团反应。用 aHNE 修饰的蛋白质与带有叠氮基和生物素基团的点击试剂发生反应，该基团由可光裂解的接头分隔开。以这种方式修饰的肽和蛋白质在链霉亲和素珠上进行亲和纯化。用低强度紫外线光解珠子，释放结合的生物素化蛋白质或肽，即经 aHNE 修饰的蛋白质或肽。采用两种策略（a）蛋白质捕获和光释放以及（b）肽捕获和光释放来富集加合蛋白或高度富集加合物的肽混合物。通过 LC-MS/MS 对链霉亲和素纯化的肽进行蛋白质组学分析，可以鉴定加合位点。通过肽捕获和光释放从人血清白蛋白中鉴定出 30 种不同的肽，揭示了蛋白质上 18 个不同的 aHNE 加合位点。蛋白质捕获和光释放表明人血浆中的 HSA 和 ApoA1 均经过 aHNE 的显着修饰。分子与细胞蛋白质组学 8：2080-2089，2009。
HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE. Molecular & Cellular Proteomics 8: 2080-2089, 2009.