亲和纯化技术联合质谱鉴定与S100A8和S100A9相互作用的蛋白

发布时间：2018-05-04 08:18

  本文选题：亲和纯化 + SA　； 参考：《中南大学学报(医学版)》2017年04期

【摘要】：目的:利用亲和纯化技术联合质谱分析筛选鉴定与S100A8和S100A9相互作用的蛋白,为探讨S100A8和S100A9在结肠炎相关性结肠癌发生发展中的作用机制提供线索。方法:构建含有S100A8和S100A9 CDS区和Flag亲和标签的真核表达载体p3×Flag-CMV-S100A8和p3×Flag-CMV-S100A9,将其转染至HEK293细胞中,48 h后收集蛋白。运用标签分离纯化S100A8和S100A9蛋白,液相质谱/质谱分析技术(liquid chromatography mass spectrometry/mass spectrometry,LC-MS/MS)鉴定S100A8和S100A9的相互作用蛋白,通过生物信息学的方法(GO分析)归纳分析相互作用蛋白,免疫共沉淀实验鉴定S100A8和S100A9的相互作用蛋白。结果:通过标签纯化联合质谱分析成功鉴定出PKM,NPM1,e IF5A等14个与S100A8相互作用的蛋白,鉴定出14-3-3ε,PKM等6个与S100A9相互作用的蛋白。对所鉴定的相互作用蛋白进行生物信息学分析,发现与S100A8和S100A9相互作用的蛋白参与糖代谢、细胞黏附、正向调控NF-κB转录因子活性、抗凋亡等在内的生物信号通路,并利用免疫共沉淀证实PKM2与S100A8和S100A9发生相互作用,14-3-3ε与S100A9发生相互作用。结论:利用免疫共沉淀结合质谱技术筛选鉴定的S100A8和S100A9相互作用蛋白PKM2和14-3-3ε,可能为阐明S100A8和S100A9在结肠炎相关性结肠癌发生发展中的作用机制提供重要线索。
[Abstract]:Objective: to screen and identify proteins interacting with S100A8 and S100A9 by affinity purification and mass spectrometry in order to provide clues to the role of S100A8 and S100A9 in the development of colitis associated colon cancer. Methods: eukaryotic expression vectors p3 脳 Flag-CMV-S100A8 and p3 脳 Flag-CMV-S100A9 containing S100A8 and S100A9 CDS regions and Flag affinity tags were constructed and transfected into HEK293 cells for 48 h to collect proteins. S100A8 and S100A9 proteins were separated and purified by label, and the interaction proteins between S100A8 and S100A9 were identified by liquid phase mass spectrometry / mass spectrometry (LC-MS / MS). The interaction proteins were analyzed by bioinformatics method (go analysis). The interaction protein between S100A8 and S100A9 was identified by immunoprecipitation assay. Results: 14 proteins interacting with S100A8 were successfully identified by tag purification and mass spectrometry, and 6 proteins interacting with S100A9 were identified, such as 14-3-3 蔚 -PKM. Bioinformatics analysis of the identified interacting proteins showed that the proteins interacting with S100A8 and S100A9 were involved in glucose metabolism, cell adhesion, positive regulation of NF- 魏 B transcription factor activity, anti-apoptosis, and so on. The interaction of PKM2 with S100A8 and S100A9 was confirmed by immunoprecipitation. 14-3-3 蔚 interacted with S100A9. Conclusion: the identification of PKM2 and 14-3-3 蔚 of S100A8 and S100A9 interaction proteins by immunoprecipitation and mass spectrometry may provide important clues for elucidating the role of S100A8 and S100A9 in the pathogenesis of colitis associated colon cancer.
【作者单位】： 中南大学湘雅三医院消化内科;中南大学湘雅三医院湖南省非可控性炎症与肿瘤重点实验室;中南大学肿瘤研究所;中南大学高等研究中心高分辨质谱实验室;
【基金】：国家自然科学基金(81472286) 湖南省自然科学基金(2016JJ6153) 中南大学博士生自主探索创新项目(2015zzts100);中南大学米塔尔学生创新项目(15MX47) 沈守荣教授的湘雅名医基金~~
【分类号】：R3411
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