ANALYSIS OF MYCOBACTERIUM ANTIGENS BY VIBRATIONALLY COOLED MALDI FTMS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。最近对分枝杆菌抗原的研究表明，cd1限制性t细胞识别的配体不是肽，而是脂质（如霉菌酸）和糖脂质（如脂阿拉伯甘露聚糖和磷脂酰肌醇甘露糖）。大多数CD1蛋白呈递的抗原的精确结构尚未被报道。cd1呈递的糖脂抗原，其结构到目前为止已经在我们的实验室和其他地方确定，是由能量不稳定键连接的多种构建块组成的，并且通常在自然界中作为密切相关结构的复杂混合物出现。BUSM MS Resource开发的薄层色谱（TLC）与振动冷却基质辅助激光解吸/电离傅里叶变换质谱（VC MALDI-FTMS）直接耦合的方法现在被应用于体内特异性生产的分枝杆菌脂质样品。cd1呈递的糖脂抗原的结构应该被揭示出来，包括碳水化合物残基的数量和它们的附着点，以及分支的精确排列和酰基链的结构。作为实施本项目的第一步，使用TLC板进行了浓度/灵敏度测试，TLC板上装有糖脂和磷脂标准品，如GMM， DDM和PI。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent studies of the Mycobacterium antigens have shown that the ligands recognized by CD1-restricted T-cells, are not peptides, but instead are lipids (e.g., mycolic acids) and glycolipids (e.g., lipoarabinomannan and phosphatidylinositol mannoside). The precise structures of the antigens presented by most CD1 proteins have not yet been reported. CD1-presented glycolipid antigens, for which structures have so far been determined in our laboratories and elsewhere, are composed of diverse building blocks linked by energetically labile bonds and often occur in nature as complex mixtures of closely related structures. The method the BUSM MS Resource has developed for direct coupling of thin layer chromatography (TLC) with vibrationally-cooled matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (VC MALDI-FTMS) is now being applied to samples of mycobacterial lipids specifically produced in vivo. The structure of CD1-presented glycolipid antigens should be revealed, with respect to the number of the carbohydrate residues and their attachment points, as well as the precise arrangement of the branching and the structure of acyl chains. As a first step in implementing this project, a test of concentration/sensitivity has been performed using TLC plates loaded with glycolipid and phospholipid standards, such as GMM, DDM, and PI.