Research Day 1st Prize-Oral Presentation

COMPREHENSIVE CHARACTERISATION OF THE CELL WALL PROTEOME OF MYCOBACTERIUM SMEGMATIS USING GEL-FREE MASS SPECTROMETRY
Clemens Hermann, Nelson C. Soares, and Jonathan M Blackburn

Division of Chemical and Systems Biology, Department of Integrative Biomedical Sciences, FHS, University of Cape Town.

Infection by Mycobacterium tuberculosis remains one of the biggest global health threats. Understanding the cell wall of mycobacteria represents one of the most important tasks to better design drugs that target mycobacteria or to identify B or T cell antigens that are suitable for vaccination strategies, but remains a challenge due to its complexity in composition. In this study, we successfully developed a gel-free approach to specifically look at cell wall proteins in Mycobacterium smegmatis. The cell wall was subjected to differential centrifugation, differential detergent solubilisation and phase separation to yield the genuine cell wall proteome. The protein extracts were digested by filter-assisted sample preparation for LC-MS/MS analysis on a Q Excative mass spectrometer and identified proteins subjected to a strict bioinformatics pipeline. This resulted in the unprecedented identification of 95 lipoproteins, 481 membrane proteins containing at least one transmembrane helix and 141 secreted proteins. Gene ontology enrichment showed that these proteins are involved in cell wall biogenesis, virulence, phosphorylation and transport. This list might represent the most comprehensive list of cell wall proteins in Mycobacterium smegmatis thus far. Our approach might provide the basis for further quantitative proteomic studies into the role of the cell wall proteome of mycobacteria in virulence or during drug exposure.