Immunoproteomics

描述（由申请人提供）：拟议的研究旨在评估使用质谱读数的新方法，以提供对选定蛋白质的敏感检测，以及监测靶向抗原翻译后修饰事件的方法。提议的检测包括一个由固定抗体（免疫）介导的抗原捕获步骤和一个涉及结合抗原（蛋白质组学）的质谱分析的分析步骤。该项目的目标是开发快速灵敏的抗原捕获方法，从不相关蛋白质的复杂混合物中捕获抗原，允许随后使用飞行时间质谱法精确测定结合抗原的分子量。区分靶抗原大小的细微变化的能力将允许对任何靶抗原的翻译后修饰事件进行分析。此外，根据质谱读出的特定分子量峰下面积获得半定量数据的能力将被严格评估。概念验证研究将集中在分泌的链球菌半胱氨酸蛋白酶SpeB的一些特性上，已知SpeB在翻译后修饰表面抗吞噬M蛋白，并降解分泌的细菌纤维溶酶原激活剂SK。

DESCRIPTION (provided by applicant): The proposed studies are designed to evaluate new methods using a mass spectral readout to provide sensitive, detection of selected proteins, as well as methods to monitor post-translational modification events of targeted antigens. The proposed assay involves an antigen capture step mediated by immobilized antibody (immuno) and an analytical step involving mass spectral analysis of bound antigen (proteomics). The goal of the project is to develop rapid sensitive methods of antigen capture from complex mixtures of unrelated proteins in a maimer that permits the subsequent precise molecular weight determination of the bound antigen using time of flight mass spectrometry. The ability to distinguish subtle variation in the size of a targeted antigen will allow analysis of post-translational modification events for any targeted antigen to be achieved. In addition, the ability to obtain semi-quantitative data based on the area under a specific molecular weight peak on the mass spectral read-out will be critically evaluated. The proof of concept studies will focus on a number of properties of the secreted streptococcal cysteine protease SpeB which is known to post-translationally modify the surface anti-phagocytic M protein, and degrade the secreted bacterial plasminogen activator SK.