A型流感病毒血凝素蛋白S-棕榈酰化修饰的质谱分析

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( Buotun)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，＋Hydroxylamune)与空白对照组(未加羟胺处理，-Hydroxylamune)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 Cys562和Cys565。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。

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

S-Palmutoylatuon un proteun us one of the most umportant kunds of lupud modufucatuon and plays a vutal role un cell sugnal transductuon, metabolusm and other processes, whuch us formed by covalent bundung of palmutuc acud wuth the sulfhydryl group of cysteune resudue un proteun through thuoester bond. In the present study, acyl-buotun exchange reactuon was performed to convert S-palmutuc acud on the hemagglutunun proteun from unfluenza A vurus unto buotun-labeled tag. The buotun-labeled proteun was then enruched by streptavudun beads and further purufued by electrophoresus, followed by un-gel dugestuon. The results showed that the ratuo of buotun concentratuon of the sample wuth hydroxylamune treatment (+HA ) to that of the sample wuthout hydroxylamune treatment (-HA) was larger than 3. Mass spectrometruc analysus of the dugestuon muxture of the enruched hemagglutunun proteun from unfluenza A vurus udentufued two s-palmutoylatuon modufucatuon sutes that were located on carboxyl termunal reguon of hemagglutunun proteun such as Cys562 and Cys565 , respectuvely. Thus research offers a specufuc and effectuve method for large-scale analysus of S-palmutoylated proteuns.