本文選題：酪氨酸磷酸化 + 富集��； 參考：《分析化學(xué)》2017年10期

【摘要】：酪氨酸磷酸化及其相應(yīng)激酶活性的研究在抗腫瘤藥物靶點的研發(fā)中具有重要意義。由于酪氨酸磷酸化僅占蛋白質(zhì)總磷酸化含量的不足0.1%,因此規(guī)�；睦野彼崃姿峄b定面臨著重大技術(shù)挑戰(zhàn)。本研究構(gòu)建了TiO_2串聯(lián)C_(18)反相填料的離心式富集裝置,結(jié)合抗體免疫沉淀法,建立了酪氨酸磷酸肽的富集策略。此新型富集裝置由吸頭、適配器和離心(EP)管組成,將TiO_2富集磷酸肽和C_(18)填料反相分離磷酸肽有機結(jié)合,以離心的方式進行樣品的上樣、清洗、洗脫和分離,再通過抗酪氨酸磷酸化抗體進一步特異性富集酪氨酸磷酸肽,從而實現(xiàn)了酪氨酸磷酸肽的高效富集和大規(guī)模質(zhì)譜鑒定。通過離心式富集裝置簡化了實驗步驟,減少了樣品損失和人為因素干擾;而且離心式、平行化的樣品處理方式可顯著提高分析通量。將此策略成功用于小鼠肝臟蛋白質(zhì)酪氨酸磷酸化肽段的富集和質(zhì)譜鑒定,在5 mg鼠肝蛋白中共鑒定出967個酪氨酸磷酸化位點,對應(yīng)545個蛋白質(zhì),顯示了其在蛋白質(zhì)組學(xué)研究中的應(yīng)用潛力。
[Abstract]:The study of tyrosine phosphorylation and its kinase activity is of great significance in the development of antitumor drug targets.Since tyrosine phosphorylation accounts for less than 0.1% of the total protein phosphorylation, the identification of tyrosine phosphorylation on a large scale is facing great technical challenges.In this study, a centrifugal enrichment device with TiO_2 series CST18) reverse phase filler was constructed, and the enrichment strategy of tyrosine phosphopeptide was established with antibody immunoprecipitation method.This new type of enrichment device consists of suction head, adapter and centrifuge EPP-tube. It organically combines TiO_2 enrichment of phosphate peptide with CST18) filler to separate phosphate peptide in reverse phase, and carries out sample preparation, cleaning, elution and separation by centrifugation.Furthermore, the tyrosine phosphopeptide was further enriched by anti-tyrosine phosphorylation antibody, so that the high concentration of tyrosine phosphopeptide and the identification of mass spectrometry were realized.The experiment procedure is simplified by centrifugal enrichment device, and the sample loss and human interference are reduced, and the analytical flux can be significantly increased by the centrifugal and parallel sample treatment.The strategy was successfully applied to the enrichment and mass spectrometry identification of the tyrosine phosphorylated peptide segment of mouse liver protein. 967 tyrosine phosphorylation sites were identified at 5 mg mouse liver protein, corresponding to 545 proteins.It shows its potential application in proteomics research.
【作者單位】： 軍事醫(yī)學(xué)科學(xué)院放射與輻射醫(yī)學(xué)研究所國家蛋白質(zhì)科學(xué)研究中心蛋白質(zhì)組學(xué)國家重點實驗室北京蛋白質(zhì)組研究中心;
【基金】：國家重點研發(fā)計劃(Nos.2013CB911204,2016YFA0501403) 國家自然科學(xué)基金(Nos.21235001,21405175,21675172)資助~~
【分類號】：O629.72;O652.6
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本文編號：1764044