基于非标记分析方法应用四极杆-静电场轨道阱-线性离子阱三合一高分辨质谱仪鉴定中国仓鼠卵巢细胞膜蛋白

doi:10.19871/j.cnki.xfcrbzz.2024.02.007

新发传染病电子杂志 ›› 2024, Vol. 9 ›› Issue (2): 34-40.doi: 10.19871/j.cnki.xfcrbzz.2024.02.007

基于非标记分析方法应用四极杆-静电场轨道阱-线性离子阱三合一高分辨质谱仪鉴定中国仓鼠卵巢细胞膜蛋白

刘雅雯^1,2, 梁雄顺¹, 胡绪乔¹, 刘小倩¹, 徐万娜¹, 洪文旭^1,2

1.深圳市慢性病防治中心中心实验室,广东深圳 518020;
2.汕头大学公共卫生学院,广东汕头 515041

收稿日期:2023-01-23 出版日期:2024-04-30 发布日期:2024-05-22
通讯作者: 洪文旭,Email:szbloodcenter@hotmail.com
基金资助:
深圳医疗卫生“三名工程”（SZSM201811057）

Identification of membrane proteins in Chinese hamster ovary cell using Orbitrap fusion lumos mass spectrometers based on non-label technique

Liu Yawen^1,2, Liang Xiongshun¹, Hu Xuqiao¹, Liu Xiaoqian¹, Xu Wanna¹, Hong Wenxu^1,2

1. Central Laboratory, Shenzhen Center for Chronic Disease Control, Guangdong Shenzhen 518020,China;
2. School of Public Health, Shantou University, Guangdong Shantou 515041, China

Received:2023-01-23 Online:2024-04-30 Published:2024-05-22

摘要/Abstract

摘要： 目的探讨应用四极杆-静电场轨道阱-线性离子阱（Orbitrap fusion lumos）三合一高分辨质谱仪鉴定中国仓鼠卵巢（Chinese hamster overy,CHO）细胞的全蛋白质组和膜蛋白质组的非标记（label-free）分析方法,并评价其应用效果,为研究膜蛋白相关传染病的发生机制提供方法学基础。方法贴壁培养中国仓鼠卵巢细胞,提取、富集和酶解处于对数生长期细胞的全蛋白和膜蛋白,用高效液相色谱仪联用四极杆-静电场轨道阱-线性离子阱三合一高分辨质谱仪对酶解后的多肽进行分析,利用Maxquant软件和Uniprot数据库进行搜库及鉴定,最后对鉴定膜蛋白进行基因本体论（gene ontology,GO）功能分类。结果全蛋白组一共检测出14 300个肽段,搜库得到2708个蛋白,其中可信蛋白2002个（FDR<1%且特异性肽段>1）;膜蛋白组一共检测出13 179个肽段,搜库得到2594个蛋白,其中可信蛋白1873个。在鉴定的全蛋白组和膜蛋白组中定量结果前20的蛋白中,全蛋白组中与膜相关的蛋白有4个,占比20%;膜蛋白组中与膜相关的蛋白有10个,占比50%。在膜蛋白组中,GO功能分类表明分子功能上主要涉及结合和催化活性,在生物学进程上主要涉及细胞进程、代谢过程等。结论膜蛋白组中与膜相关的蛋白显著高于本实验的全蛋白组,基于非标记分析方法应用Orbitrap fusion lumos的分析技术是鉴定细胞膜蛋白的有效方法。

关键词: 中国仓鼠卵巢细胞, 膜蛋白质, 非标记方法, 四极杆-静电场轨道阱-线性离子阱高分辨质谱仪

Abstract: Objective To explore the method of identifying the whole proteome and membrane protein of Chinese hamster ovary (CHO) cells by using the Orbitrap fusion lumos mass spectrometer based on label-free technique,and evaluate its application effect. It provides the methodological basis for studying the pathogenesis of membrane protein-related infectious diseases. Method CHO cells were cultured, and then the whole protein and membrane protein of the cells in the logarithmic growth phase were extracted, enriched and enzymatically hydrolyzed. The peptides were identified by HPLC-Orbitrap fusion lumos three in mass spectrometer. Maxquant software and Uniprot database were used for database search and identification. Finally, the identified membrane proteins were classified by GO function. Result A total of 14 300 peptide segments were detected in the whole proteome, and 2708 proteins were found in the database search,including 2002 credible proteins(FDR<1% and unique peptide>1); A total of 13 179 peptide segments were detected in the membrane proteome, and 2594 proteins were found in the database search,including 1873 credible proteins. Among the top 20 proteins in the identified total protein and membrane protein groups, there are 4 membrane related proteins in the total protein group, accounting for 20%; There are 10 membrane related proteins in the membrane proteome, accounting for 50%. In the membrane protein components. GO function classification shows that molecular function is mainly related to binding and catalytic activity, while in biological processes, it mainly involves cellular processes, metabolic processes. Conclusion The membrane-associated proteins in the membrane protein group were significantly higher than those in the whole protein group,and the application of Orbitrap fusion lumos analysis technology based on label-free technique is an effective method for identifying cell membrane proteins.

Key words: Chinese hamster ovary cells, Membrane protein, Non labeling method, Orbitrap fusion high-resolution mass spectrometer

中图分类号:

刘雅雯, 梁雄顺, 胡绪乔, 刘小倩, 徐万娜, 洪文旭. 基于非标记分析方法应用四极杆-静电场轨道阱-线性离子阱三合一高分辨质谱仪鉴定中国仓鼠卵巢细胞膜蛋白[J]. 新发传染病电子杂志, 2024, 9(2): 34-40.

Liu Yawen, Liang Xiongshun, Hu Xuqiao, Liu Xiaoqian, Xu Wanna, Hong Wenxu. Identification of membrane proteins in Chinese hamster ovary cell using Orbitrap fusion lumos mass spectrometers based on non-label technique[J]. Electronic Journal of Emerging Infectious Diseases, 2024, 9(2): 34-40.

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基于非标记分析方法应用四极杆-静电场轨道阱-线性离子阱三合一高分辨质谱仪鉴定中国仓鼠卵巢细胞膜蛋白

Identification of membrane proteins in Chinese hamster ovary cell using Orbitrap fusion lumos mass spectrometers based on non-label technique

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