中国中药杂志

2021, v.46(24) 6422-6434

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基于nano LC-Q-Exactive-MS/MS技术分析阿胶中的蛋白多肽类物质
Analysis of peptides and proteins from Asini Corii Colla using nano LC-Q-Exactive-MS/MS

褚夏燕;孙梦茹;贾贵华;王延涛;顾建军;刘海滨;葛广波;
CHU Xia-yan;S UN Meng-ru;JIA Gui-hua;WANG Yan-tao;GU Jian-jun;LIU Hai-bin;GE Guang-bo;Instituteof Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine;National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Dong-E-E-Jiao Co., Ltd.;School of Pharmaceutical Sciences,Jilin University;

摘要(Abstract):

基于鸟枪法(shotgun)蛋白质组学分析技术对东阿阿胶成品中的蛋白多肽类物质进行系统分析。采用高pH反相色谱预分离的方法对酶解后的阿胶蛋白多肽类物质进行分级;采用Thermo Scientific EASY column预柱(100μm×2 cm, 5μm, C_(18))及Thermo Scientific EASY column分析柱(75μm×100 mm, 3μm, C_(18))为固定相对酶解后的阿胶蛋白多肽类物质进行有效的分离,以0.1%甲酸乙腈水溶液(乙腈为84%)-0.1%甲酸水为流动相进行梯度洗脱;借助nano LC-Q-Exactive-MS/MS分析技术在正离子模式下采集阿胶多肽组的质谱数据。通过MaxQuant软件结合马属蛋白数据库、质谱数据分析及参考相关文献,从成品东阿阿胶中共分离鉴定出2 291个马属肽段,同时首次从阿胶成品中发现了255个含有美拉德修饰(AML、CML、CEL)的马属肽段;并借助数据库搜寻对上述肽段的蛋白归属进行对比分析,发现所检出的肽段源于678个马属蛋白。该研究借助nano LC-Q-Exactive-MS/MS分析技术和鸟枪法蛋白质组鉴定法实现了阿胶成品中的蛋白多肽类物质快速、准确分析,为深入了解阿胶的化学物质组和效应物质提供了新的线索和关键信息。
This paper aims to systematically analyze the peptides and proteins from Asini Corii Colla(ACC) through shotgun proteomics. After high-pH reversed-phase fractionation, the proteins and peptides in the hydrolysate of ACC were further separated by nano LC-Q-Exactive-MS/MS under the following conditions: Thermo Scientific EASY column(100 μm×2 cm, 5 μm, C_(18)) as precolumn, Thermo Scientific EASY column(75 μm×100 mm, 3 μm, C_(18)) for solid phase extraction, gradient elution with 0.1% formic acid in water(mobile phase A) and 84% acetonitrile in water containing 0.1% formic acid(mobile phase B), and MS in positive ion mode. Based on Uniprot_Equus caballus, MS data, and literature, 2 291 peptides were identified from ACC by MaxQuant, with 255 Maillard reactions(AML, CML, CEL)-modified peptides identified for the first time. Through alignment, the peptides were found to belong to 678 equine proteins. In conclusion, the combination of nano LC-Q-Exactive-MS/MS and shotgun proteomics achieved rapid and accurate identification of the proteins and peptides in ACC, which provides the key information and new insights for further investigation of chemicals and effective substances in ACC.

关键词(KeyWords): 阿胶;蛋白多肽类;美拉德修饰多肽;高pH反相分级;shotgun蛋白组分析
Asini Corii Colla;proteins and peptides;Maillard reactions-modified peptides;high-pH reversed-phase fractionation;shotgun proteomics

Abstract:

Keywords:

基金项目(Foundation): 泰山产业领军人才项目(TSCY20180234);; 国家重点研发计划“中医药现代化研究”重点专项(2017YFC1700200,2017YFC1702000);; 山东省重点研发计划重大科技创新工程项目(2019JZZY020907)

作者(Author): 褚夏燕;孙梦茹;贾贵华;王延涛;顾建军;刘海滨;葛广波;
CHU Xia-yan;S UN Meng-ru;JIA Gui-hua;WANG Yan-tao;GU Jian-jun;LIU Hai-bin;GE Guang-bo;Instituteof Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine;National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Dong-E-E-Jiao Co., Ltd.;School of Pharmaceutical Sciences,Jilin University;

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