Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses
Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses
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DOI：
10.1021/ac010081x
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发表时间：
2001-06-01
影响因子：
7.4
通讯作者：
Smith, RD
中科院分区：
文献类型：
--
作者：
Goshe, MB;Conrads, TP;Smith, RD
II方法利用结合稳定同位素和生物素标记的磷蛋白同位素编码亲和标记(PhIAT)来丰富和定量测量蛋白质O-磷酸化状态的差异。PhIAT标记方法包括氢氧离子介导的O-磷酸部分的B消除和添加含有四个烷基氢(EDT-D-0)或四个烷基氚(EDT-D-4)的1，2-乙二硫醇，然后使用(+)-biotinyl-iodoacetamidyl-3，6-dioxaoctanediamine.对EDT-D-0/D-4部分进行生物素化合成了一种含有亲核巯基和共价连接到生物素部分的同位素标记的PhIAT试剂，它具有将O-磷酸化衍生化减少到一步过程的潜在用途。利用模型磷化蛋白β-酪蛋白初步证明了PhIAT标记方法，经蛋白质降解后，用固定化亲和素亲和分离PhIAT标记的多肽，并用毛细管反相液相色谱-质谱仪进行分析，分离和鉴定了与含有已知O-磷酸化位点的多肽相对应的PhIAT标记的β-酪蛋白多肽。PhIAT标记方法也被应用于酵母蛋白提取物。PhIAT标记技术为定量测量蛋白质O-磷酸化状态的差异提供了可靠的方法。
ii method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated B-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D-0) or four alkyl deuteriums (EDT-D-4) followed by biotinylation of the EDT-D-0/D-4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, nas synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta -casein, After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase Liquid chromatography-mass spectrometry, phIAT-labeled beta -casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied to a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.