微量样本泛素化蛋白质组学研究前处理方法优化  

Optimization of pretreatment methods for ubiquitination proteomics of micro⁃samples

作　　者：梁明秀 邢美宁 黄东莹 梁石芳 应万涛 高晓霞 LIANG Mingxiu;XING Meining;HUANG Dongying;LIANG Shifang;YING Wantao;GAO Xiaoxia(School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou 510006,China;Institute of Bioomics,Academy of Military Medicine,Academy of Military Sciences,State Key Laboratory of Medical Proteomics,Beijing Proteome Research Center,National Center for Protein Sciences(Beijing),Beijing Institute of Lifeomics,Beijing 102206,China)

机构地区：[1]广东药科大学药学院,广东广州510006 [2]军事科学院军事医学研究院生命组学研究所,医学蛋白质组全国重点实验室,北京蛋白质组研究中心,国家蛋白质科学中心(北京),北京102206

出　　处：《海南医学院学报》2024年第17期1321-1326,共6页Journal of Hainan Medical University

基　　金：国家重点研发计划课题(2021YFA1300201)。

摘　　要：目的:对影响泛素化肽段富集的因素进行系统评估,以提升基于串联质谱的蛋白质组学方法检测微量样本泛素化修饰位点的能力,增加泛素化修饰鉴定覆盖度,进而为微量临床样本的泛素组学研究提供参考方法。方法:本研究中运用100μg HeLa全蛋白酶解肽段系统评估了在泛素化富集过程中不同抗体用量(9.375μg、15.625μg、31.25μg)、不同富集体积(300μL、600μL、1000μL)中鉴定泛素化肽段和位点的能力,以及对比冻干前加入二甲亚砜能否提升泛素化肽段鉴定能力。结果:不同富集条件对泛素化肽段的富集和鉴定效率有所不同。在不同的抗体输入量的评估中,使用15.625μg抗体珠能使富集效率达到57%且获得较理想的泛素化肽和位点鉴定量;在不同体积的对比中,使用600μL富集缓冲液能使单针检测出的泛素化肽段和位点超过10000且两次重复实验泛素化位点的定量相关系数达到0.95;冻干前加入二甲亚砜提高了疏水肽段的鉴定能力,使泛素化位点增加4.59%。结论:通过系统优化,对于100μg肽段的富集推荐使用15.625μg抗体量以及600μL富集体积,在样本冻干前加入二甲亚砜可以有效增加疏水性肽段的鉴定。Objective:To enhance the ability of tandem mass spectrometry based⁃proteomics methods for detecting ubiquitina⁃tion modification sites in trace samples and increase the coverage of ubiquitination modification identification.This paper systemati⁃cally evaluates the factors affecting the enrichment of ubiquitination peptides,hoping to provide a reference method for the study of ubiquitination modification proteomics in trace clinical samples.Methods:In this study,we used 100μg HeLa protein digest pep⁃tides to systematically evaluate the effects of different conditions on enrichment and identification ability of ubiquitinated peptides and sites.During ubiquitination enrichment,the ability to identify ubiquitinated peptides and sites was investigated at different anti⁃bodies dosages(9.375μg,15.625μg,and 31.25μg)and enrichment volumes(300μL,600μL,and 1000μL),and the effect of adding dimethyl sulfoxide before lyophilization on the improvement of ubiquitinated peptide identification ability was also investigat⁃ed.Results:The enrichment and identification efficiency of ubiquitination peptides varied with different enrichment conditions.By evaluating the amount of antibody input,15.625μg bead⁃conjugated antibodies result in the best enrichment efficiency,up to 57%,and better identification quantities in both ubiquitinated peptides and sites.By comparing the effects of enrichment volumes,600μL enrichment buffer resulted in more than 10000 ubiquitination peptides and sites in a single injection,and the quantitative correlation coefficient of the ubiquitinated sites in the two replicate experiments reached 0.95.The addition of dimethyl sulfoxide before lyophilization improved the identification capacity of hydrophobic peptides,resulting in an increase of 4.59%in the identi⁃fied ubiquitination sites.Conclusion:After systematic optimization,15.625μg of bead⁃conjugated antibodies and 600μL system volumes were recommended for the enrichment of 100μg peptides,and the addition of dimethyl sulfoxide be

关 键 词：泛素化 富集 DMSO LC-MS/MS 

分 类 号：TQ937[化学工程] R313[医药卫生—基础医学]