利用質譜技術鑑定甲型流感病毒血球凝集素H7蛋白的雙硫鍵鍵結

Abstract

雙硫鍵為影響蛋白質功能與活性的關鍵結構因子，對於可作為疫苗的重組抗原蛋白來說，雙硫鍵會影響其摺疊以及結構，錯接的雙硫鍵會嚴重的影響抗原與抗體結合的特異性，因此蛋白質正確雙硫鍵鍵結的鑑定是極重要的。本篇研究中，我們利用雙甲基化反應搭配液相層析質譜技術以及RADAR軟體的輔助，鑑定甲型流感病毒H7N9病毒顆粒以及其重組的血球凝集素H7蛋白： H7蛋白三聚體、H7蛋白單體以及Dsbc-H7、Dsbc-HA1。蛋白質樣品利用不同的酵素組合在酸性環境下水解，在本研究中使用包含: 胰蛋白酶 (trypsin)、trypsin + chymotrypsin、trypsin + asp-n等組合。使用trypsin + asp-n的酵素組合可在全部的樣品中鑑定到四個雙硫鍵鍵結，此為鑑定H7蛋白最佳的酵素組合。雙硫鍵片段C272-C296可在H7N9樣品中被鑑定到，而雙硫鍵片段C4-C458則在H7-trimer中被鑑定到。此雙硫鍵分析方法可對於蛋白質活性提供重要的資訊，據我們所知，此篇為首次利用雙硫鍵分析的方法探討甲型流感病毒H7N9血球凝集素H7重組蛋白活性的研究。Disulfide linkage is a crucial structure factor that affect the functionality and activity of proteins. As to the recombinant antigen proteins used for vaccine production, disulfide bonds affect their folding and structure. Disulfide bond scrambling would greatly influence the specificity of antigen-antibody binding. Thus, confirmation of the correct disulfide bond pairings is important. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was used to identify the disulfide bonds in influenza A virus particle H7N9 and its recombinant H7 proteins, including H7-trimer, H7-monomer, Dsbc-H7, and Dsbc-HA1. They were digested with different protease combinations including trypsin, trypsin/chymotrypsin, and asp-n in acidic condition. Four disulfide bonds were identified in all samples using trypsin plus asp-n, which gave the most disulfide bond identifications among all protease combinations. The disulfide linkage peptides, C272-C296 and C4-C458, can be identified only in H7N9 virus particle and H7-trimer, respectively. Eventually, this proposed disulfide linkage analysis can provide significant knowledge for protein activity study. To our knowledge, this is the first report using LC-MS/MS for the analysis of disulfide linkages on influenza A virus hemagglutinin H7 protein.