ANALYSIS OF MYCOBACTERIUM ANTIGENS BY VIBRATIONALLY COOLED MALDI FTMS

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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.