A型流感病毒血凝素蛋白S-棕榈酰化修饰的质谱分析

Mass Spectrometric Analysis of S-Palmitoylation of Hemagglutinin from Influenza A Virus

蛋白质S-棕榈酰化修饰是指棕榈酸分子通过硫酯键共价结合在蛋白质分子的半胱氨酸(S)的巯基侧链上,是蛋白质脂类修饰的重要形式之一,在细胞信号转导、代谢等过程中起着重要作用。本实验首先利用酰基-生物素置换反应,将A型流感病毒血凝素蛋白上的S-棕榈酸分子转换为含有生物素(Biotin)分子的标签。生物素标记蛋白经特异性富集、电泳分离纯化后,进行胶内水解。再利用质谱技术对水解混合物进行分析。结果表明,经酰基-生物素置换方法处理A型流感病毒裂解产物后,蛋白质耦联生物素的浓度(羟胺处理,+Hydroxylamine)与空白对照组(未加羟胺处理,-Hydroxylamine)的比值大于3;对经富集后的流感病毒血凝素蛋白进行了质谱分析,鉴定了A型的两个S-棕榈酰化修饰位点,分别位于蛋白羧基末端的Cys_(562)和Cys_(565)。本研究为大规模研究S-棕榈酰化修饰蛋白提供了一种特异、有效的分析方法。

S-Palmitoylation in protein is one of the most important kinds of lipid modification and plays a vital role in cell signal transduction,metabolism and other processes,which is formed by covalent binding of palmitic acid with the sulfhydryl group of cysteine residue in protein through thioester bond. In the present study,acyl-biotin exchange reaction was performed to convert S-palmitic acid on the hemagglutinin protein from influenza A virus into biotin-labeled tag. The biotin-labeled protein was then enriched by streptavidin beads and further purified by electrophoresis,followed by in-gel digestion. The results showed that the ratio of biotin concentration of the sample with hydroxylamine treatment（ ＋ HA） to that of the sample without hydroxylamine treatment（-HA） was larger than 3. Mass spectrometric analysis of the digestion mixture of the enriched hemagglutinin protein from influenza A virus identified two s-palmitoylation modification sites that were located on carboxyl terminal region of hemagglutinin protein such as Cys_（562） and Cys_（565）,respectively. This research offers a specific and effective method for large-scale analysis of S-palmitoylated proteins.