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Orbitrap Exploris 480质谱在定量蛋白质组学应用中的优化和评测
引用本文:周岳,杨湘云,黄敏,李想,关锋.Orbitrap Exploris 480质谱在定量蛋白质组学应用中的优化和评测[J].生物化学与生物物理进展,2021,48(2):214-226.
作者姓名:周岳  杨湘云  黄敏  李想  关锋
作者单位: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 200120,2)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 710069,3)西北大学,生命科学学院,西安 710069
基金项目: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...

关 键 词:定量蛋白质组学  非标记定量  TMT  FAIMS  Orbitrap  Exploris  480
收稿时间:2020/7/20 0:00:00
修稿时间:2020/8/20 0:00:00

Optimization and Evaluation of Orbitrap Exploris 480 Mass Spectrometry for Quantitative Proteomics
ZHOU Yue,YANG Xiang-Yun,HUANG Min,LI Xiang and GUAN Feng.Optimization and Evaluation of Orbitrap Exploris 480 Mass Spectrometry for Quantitative Proteomics[J].Progress In Biochemistry and Biophysics,2021,48(2):214-226.
Authors:ZHOU Yue  YANG Xiang-Yun  HUANG Min  LI Xiang and GUAN Feng
Institution:1)Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China,2)Themofisher Scientific, Shanghai 200120, China,2)Themofisher Scientific, Shanghai 200120, China,3)The College of Life Sciences, Northwest University, Xi''an 710069, China,3)The College of Life Sciences, Northwest University, Xi''an 710069, China
Abstract: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, single-cell proteomics, plasma proteomics and yeast proteomics. The results showed that the collision energy of 27, the resolution of the fragment spectrum of 15K, 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 1 259 and 1 725 proteins in 200 pg and 500 pg of HeLa cell lysate, respectively, achieving deep coverage of single-cell proteomics. In FAIMS DDA experiment, we chose CV-45V for 60 min or 90 min gradient, CV-45V-65V combinations for 120 min or 150 min gradient to obtain the optimal protein identifications, and identified 6 300, 6 994 and 7 500 proteins in 60 min, 120 min and 150 min from 293T proteome, respectively. In FAIMS DIA experiment, we used CV-45V-65V voltage sweeping, 60 isolation windows, and obtained the optimal proteins identifications and quantitative reproducibility. We quantified 7 019 proteins in 293T cell lysate and 1 077 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. 10 989 peptides and 2 162 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.
Keywords:quantitative proteomics  label-free quantitative  TMT  FAIMS  Orbitrap Exploris 480
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