尺寸排阻-反相液相色谱-质谱联用技术在大鼠肾脏翻译后修饰蛋白质鉴定中的应用  

Size exclusion-reverse liquid column chromatography-mass spectrometry and its application in the identification of post-translationally modified proteins in rat kidney

作　　者：李健民 卓越 张毅达 李娜 伍建林 LI Jianmin;ZHUO Yue;ZHANG Yida;LI Na;WU Jianlin(Faculty of Chinese Medicine, State Key Laboratory of Quality Research in Chinese Medicine, Macao University of Science and Technology, Macao 999078, China)

机构地区：[1]澳门科技大学中医药学院,中药质量研究国家重点实验室,中国澳门999078

出　　处：《色谱》2021年第1期87-95,共9页Chinese Journal of Chromatography

基　　金：澳门特别行政区科学技术发展基金会(0090/2018/A3).

摘　　要：LC-MS联用技术在蛋白质组学研究中具有重要的作用,但是在复杂的生物体系中,由于样品的高度复杂性和其中蛋白质含量的巨大差异,执行全面且无倾向的蛋白质组分析是一项挑战。因此,在液相色谱分离中采用基于不同原理的色谱分离方法来降低蛋白质样本的复杂度,并对微量蛋白质进行富集,对后续采用质谱方法进行信息的采集和深入分析至关重要。在这里我们开发了一种基于尺寸排阻色谱(SEC)与反相液相色谱(RPLC)结合的新方法来进行复杂体系蛋白质的分离和鉴定,特别是对于微量蛋白质的分析。首先使用SEC对蛋白质进行分离和富集,并酶解成多肽,再通过RPLC-MS联用的方法对酶解后的多肽进行分离和鉴定。结果显示使用上述方法可以有效降低蛋白质样本的复杂度,并有效提高微量蛋白质的鉴定能力,可从大鼠肾脏鉴定出23621个肽段及1345个蛋白质,比常规的二维强阳离子交换-反相液相色谱法(2D SCX-RPLC)鉴定到的肽段及蛋白质分别多出69%及27%。此外,该方法对肾脏翻译后修饰(PTM)蛋白质的鉴定显示出更多的优势,翻译后修饰的多肽鉴定率显著增加,特别是磷酸化肽段的鉴定效率可达到靶向富集策略的水平。在此展示的SEC-RPLC-MS可以更好地了解蛋白质翻译后修饰对肾脏的影响,最终将有助于增加我们对正常的生理性肾功能以及病理过程机制的理解。Proteomics is an emerging field that has been shown to play a crucial role in unveiling the mechanisms underlying physiological and pathological processes,and liquid chromatography-mass spectrometry(LC-MS)is one of the most important methods employed in this field.However,in complex biological systems,such as eukaryotes,it is challenging to perform a comprehensive and unbiased proteome analysis due to the high complexity of biological samples and enormous differences in sample contents.For example,post-translational modifications(PTMs)in proteins are imperative for cell signaling,but post-translationally modified proteins account for about 1%of the total proteins in a single cell,making their identification extremely difficult.Therefore,chromatographic separation methods based on different principles are generally applied to reduce the complexity of biological samples and enrich trace proteins for their identification through mass spectrometry(MS).In this study,we developed a new proteomics method by combining size exclusion chromatography(SEC)and reversed-phase chromatography(RPLC),to separate and identify trace proteins in complex systems.SEC was used to separate and enrich kidney-specific proteins.After optimization of the method,it was found that 30 mmol/L of ammonium acetate could efficiently separate rat kidney proteins from the total protein fraction so that they could be eluted based on their relative molecular mass.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)analysis and LC-MS results showed that our SEC separation method not only refined the protein composition of the biological sample but also enhanced the relative contents of trace proteins through multiple injections.The collected protein fractions were further concentrated through ultrafiltration centrifugation followed by freeze-drying,which further improved the recovery of trace proteins by approximately 90%and largely decreased the time required with the use of freeze-drying alone.Thereafter,five protein fractions were sep

关 键 词：蛋白质组学 尺寸排阻色谱 翻译后修饰 

分 类 号：O658[理学—分析化学]