Abstract
Mycobacterium tuberculosis, the causative agent of the human disease Tuberculosis, utilizes the ESX-1 alternative secretion system to manipulate host-cell response to infection. Despite its importance in virulence, this system is poorly characterized and has few defined substrates.  There are at least four duplicate loci of this system in the M. tuberculosis genome, further increasing the difficulty in defining the nature of protein secretion. Previous efforts to map the secreted proteome have been unsuccessful because of the inability to define true secreted substrates from those that are present due to autolysis or variability in sample preparation. Using a novel application of a quantitative mass spectrometric technique employing iTRAQ™ reagents applied to culture supernatants from wild-type M. tuberculosis and ESX-1 mutants, we established and quantified secreted substrates from M. tuberculosis. Using this technique, we demonstrated that many of the paralogs are secreted in the absence of functional ESX-1, suggesting the presence of additional uncharacterized alternative secretion systems.  The specific challenge in quantification of these near-identical isoforms was aided by the use of a new protein search algorithm.  Using these techniques, we further defined the total secreted proteome of M. tuberculosis including the post-translational modification of secreted substrates. Additionally, we observed a quantitative increase in the transport of Sec- mediated substrates in the absence of the ESX-1 secretion system, suggesting cross talk between general and alternative secretion systems.  Recent results utilizing Multiple Reaction Monitoring (MRM) assays as Western-blot surrogates support a model that suggests substrates are independently targeted for secretion.  Results from these studies suggest that molecular proteomics can play an important role in establishing models for molecular mechanisms.
 
(1)     Champion PA et al., Science 2006 Sept 15;313(5793):1632-6.
(2)     Shilov IV, et al., MCP May-2007 Epub in-print.

Background
Matthew Champion received his B.S. Degree in Microbiology from the University Of Iowa and his Ph.D. in Biochemistry from Texas A&M University in the laboratory of Dr. James C. Hu.  In 2003, he joined the applications group at Applied Biosystems.  His research interests focus on development of quantitative LS/MS/MS approaches in proteomics, protein characterization and novel applications for characterizing virulence determinants in pathogenic Mycobacterium.