基于SPR-MS联合法鉴定Mas的互作蛋白

Identification of Mas Partner Proteins Based on the SPR-MS Coupling Technique

目的基于表面等离子共振(SPR)和质谱(MS)的联用技术从细胞裂解液中鉴定Mas互作蛋白。方法将重组蛋白GST-Mas-CT和GST分别高水平固定在2张CM5芯片的4个通道上,将细胞裂解液(总蛋白质量浓度0.5 mg·mL-1)注入芯片表面,回收蛋白经超滤辅助样品制备(FASP)后,进行MS鉴定。使用韦恩图分析Mas-CT和GST的差异互作蛋白,利用DAVID数据库对Mas-CT的差异互作蛋白进行GO和KEGG分析。结果回收蛋白中共有785个Mas-CT的差异互作蛋白;生物信息学分析显示Mas互作蛋白主要富集在免疫反应、钙离子应答、感染、血小板激活等生物学过程或通路。结论SPR-MS联合法适用于从细胞裂解液等复杂体系中捕获和鉴定靶蛋白的互作蛋白。

Objective To optimize a surface plasmon resonance(SPR)-mass spectrometry(MS)coupling technique for the identification of Mas partner proteins in cell lysates.Methods The recombinant proteins GST-Mas-CT and GST were immobilized at a high level on 4 channels of 2 CM5 chips,respectively;cell lysates(total protein concentration:0.5 mg·mL-1)were injected over the sensor surface;the recovered binding proteins were prepared using filter-assisted sample preparation(FASP)and identified using MS method;the differential interacting proteins were analyzed with Venn Diagram,and the GO and KEGG analyses were performed by DAVID database.Results A total of 785 Mas-CT differential interacting proteins were found in the recovered proteins;bioinformatics analysis showed that Mas partner proteins were primarily involved in biological processes or pathways,such as immune response,calcium ion response,infection,and platelet activation.Conclusions The optimized SPR-MS coupling technique can be utilized for fishing and identifying partner proteins of target proteins from complex systems such as cell lysates.