Characterization of PTMs on Env to support HIV vaccine development

描述(由申请人提供)：本提案的最终目标是开发广泛适用的方法来分析糖蛋白的糖基化和二硫键，并将这些方法应用于HIV包膜蛋白(Env)。这项工作将通过完成四个具体目标来完成：(1)开发一种新的糖蛋白组学方法，该方法在一次实验中量化糖基化位点占有率并同时表征糖形式异质性。(2)用Env糖基化位点占有率和糖肽异质性来回答几个重要的生物学问题。(3)开发一种用于鉴定二硫键连接多肽的自动化分析方法。(4)表征Env的二硫键，以回答几个重要的生物学问题。大部分工作将通过结合生物样品处理、高效液相色谱和质谱学(MS)数据采集和分析方面的专业知识来完成，以开发必要的工具。所有这些工具的效用将通过将它们纳入解决与艾滋病毒疫苗开发有关的问题而得到证明。特别值得注意的是AIM 2中的研究，这些工具被用来将多糖含量的变化与疫苗效力的变化联系起来。这项工作可以通过提供关于翻译后修改如何影响艾滋病毒候选疫苗效力的基本见解来广泛影响人类健康。同样，糖蛋白和二硫化物分析方法使生物分析技术能够用于从蛋白质结构/功能分析到药物开发的各种场合。与HIV蛋白质相关的应用工作是与杜克大学医学中心的Barton F.Haynes博士和哈佛大学医学院的Bing Chen博士合作完成的。

DESCRIPTION (provided by applicant): The ultimate goal of this proposal is to develop broadly applicable methods to analyze glycosylation and disulfide bonding of glycoproteins and to apply those methods to the HIV Envelope protein (Env). This work would be accomplished by completing four specific aims: (1) Develop a new glycoproteomic method, where glycosylation site occupancy is quantified and glycoform heterogeneity is simultaneously characterized, all in one experiment. (2) Profile Env glycosylation site occupancy and glycopeptide heterogeneity to answer several important biological questions. (3) Develop an automated analysis protocol for identifying disulfide- linked peptides. (4) Characterize the disulfide bonding of Env to answer several important biological questions. The bulk of the work would be completed by combining expertise in biological sample handling, HPLC, and mass spectral (MS) data acquisition and analysis to develop the necessary tools. The utility of all the tools would be demonstrated by incorporating them to solve problems relevant to HIV vaccine development. Of particular note are studies in Aim 2, where the tools are used to correlate changes in glycan content with changes in vaccine efficacy. This work can broadly impact human health by providing fundamental insights into how post-translational modifications impact HIV vaccine candidates' efficacy. Likewise, methods for glycoprotein and disulfide analysis are enabling bioanalytical technologies that can be used in a variety of venues, from protein structure/function analyses to pharmaceutical development. The application work related to HIV proteins is done in collaboration with Dr. Barton F. Haynes at Duke University Medical Center and Dr. Bing Chen at Harvard University Medical School.