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）。这项工作将完成四个具体目标：(1)开发一种新的糖蛋白组学方法，其中糖基化位点占用是量化的，同时表征糖型异质性，所有在一个实验中完成。(2)分析环境糖基化位点占用和糖肽异质性，以回答几个重要的生物学问题。(3)开发一种识别二硫键肽的自动分析方案。(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、HPLC和质谱（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，其中使用工具将多糖含量的变化与疫苗效力的变化联系起来。这项工作通过提供关于翻译后修饰如何影响HIV候选疫苗效力的基本见解，可以广泛地影响人类健康。同样，糖蛋白和二硫分析方法使生物分析技术可以用于各种场合，从蛋白质结构/功能分析到药物开发。与HIV蛋白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.