Orbitrap Exploris 480质谱在定量蛋白质组学应用中的优化和评测  

Optimization and Evaluation of Orbitrap Exploris 480 Mass Spectrometry for Quantitative Proteomics

作　　者：周岳 杨湘云 黄敏 李想 关锋 ZHOU Yue;YANG Xiang-Yun;HUANG Min;LI Xiang;GUAN Feng(Key laborutry of Carlohydrate Chemistry&Bioechrology,Mimistry of Eeducatim,School of Biotechrology,Jiangnan Uiniverity,Wuxi 214122,China;Themofisher Scientific,Shanghai 200120,China;The College of Life Sciences,Northwest Unirersity,Xi'an 710069,China)

机构地区：[1]江南大学生物工程学院糖化学与生物技术教育部重点实验室,无锡214122 [2]赛默飞世尔科技(中国)有限公司,上海200120 [3]西北大学生命科学学院,西安710069

出　　处：《生物化学与生物物理进展》2021年第2期214-226,共13页Progress In Biochemistry and Biophysics

基　　金：国家科技重大专项(2018ZX10302205-003)资助项目。

摘　　要：基于数据依赖的扫描模式(data-dependent acquisition,DDA)和数据非依赖的扫描模式(data-independent acquisition,DIA)的非标记定量(label-free quantitative,LFQ)和同位素标记TMT(tandem mass tag)定量是蛋白质组学定量中较常见的技术.本文利用最新的Orbitrap Exploris 480质谱,优化了DDA、FAIMS DDA、FAIMS DIA的非标记定量方法以及TMT定量策略的关键质谱参数,并将其应用在人细胞蛋白质组、单细胞蛋白质组、血浆蛋白质组和酵母蛋白质组分析.结果表明,在DDA实验中,设置碰撞能量为27、二级谱图的分辨率为15 K、最大离子注入时间为22 ms是最佳的参数组合.针对极微量样品200 pg~5 ng,可以根据样品量相应设置最佳的质谱参数.使用200 pg和500 pg的HeLa细胞样品,分别鉴定到1259和1725个蛋白质,从而实现了单细胞蛋白质组学的深度覆盖.在FAIMS DDA实验中,60 min或90 min梯度时选择CV-45V的补偿电压,120 min或150 min梯度时选择CV-45V-65V补偿电压组合,可以获得最佳的蛋白质鉴定结果.从293T蛋白质组中分别在60、120和150 min中鉴定6300、6994和7500个蛋白质.在FAIMS DIA实验中,使用CV-45V-65V双补偿电压切换,60个隔离窗口来获得最佳的蛋白质鉴定数目和定量重现性.在60 min内分别在293T细胞和去除高峰度的血浆中定量到7019个细胞蛋白和1077个血浆蛋白.在TMT定量实验中设置APD开启、"Precursor Fit"阈值70和Turbo TMT功能的组合同时增加了TMT定量蛋白质的数量和TMT定量的准确性.在TMT11-plex标记的酵母蛋白质组中60 min即可定量10989个多肽和2162个蛋白质.综上所述,本研究中优化的多种非标记定量和TMT定量方法为Orbitrap Exploris 480在蛋白质组学更广泛的应用提供了参考.Data-dependent acquisition(DDA)and data-independent acquisition(DIA)based label-free quantification(LFQ)and tandem mass tag(TMT)based isotope labeling quantification are two key techniques for quantitative proteomics.Here we optimized the latest Orbitrap Exploris 480 mass spectrometer parameters in DDA,FAIMS DDA,FAIMS DIA based LFQ and TMT,and show its performance in cell line proteomics,singlecell proteomics,plasma proteomics and yeast proteomics.The results showed that the collision energy of 27,the resolution of the fragment spectrum of 15 K,and maximum ion injection time of 22 ms are the best parameter combination for DDA experiment.For the proteomic analysis of ultra-low samples ranging from 200 pg-5 ng,the individual mass spectrometer parameters should be considered.We identified 1259 and 1725 proteins in 200 pg and 500 pg of He La cell lysate,respectively,achieving deep coverage of single-cell proteomics.In FAIMS DDA experiment,we chose CV-45 V for 60 min or 90 min gradient,CV-45 V-65 V combinations for 120 min or 150 min gradient to obtain the optimal protein identifications,and identified 6300,6994 and 7500 proteins in 60 min,120 min and 150 min from 293 T proteome,respectively.In FAIMS DIA experiment,we used CV-45 V-65 V voltage sweeping,60 isolation windows,and obtained the optimal proteins identifications and quantitative reproducibility.We quantified 7019 proteins in 293 T cell lysate and 1077 proteins in depleted plasma in 60 min gradient.The combination of APD on,"Precursor Fit"threshold of 70 and Turbo TMT could simultaneously increase the number of protein identifications and the accuracy of TMT quantification.10989 peptides and 2162 protein were quantified in TMT11-plex labeled yeast proteome.Taken together,the various LFQ methods and TMT quantification method optimized in this study showed high performance and various applications in quantitative proteomics.

关 键 词：定量蛋白质组学 非标记定量 TMT FAIMS Orbitrap Exploris 480 

分 类 号：O658[理学—分析化学] Q51[理学—化学]