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In addition to the general secretion pathway, Mycobacterium tuberculosis utilizes an alternative secretion apparatus called ESX-1, which is essential for control of host-cell responses to infection. Despite its importance in virulence, this system is poorly characterized and has few known substrates. There are five duplicate loci of the ESX-1 system in the genome, further increasing the difficulty in defining the nature of substrate secretion. Previous efforts to map the secreted proteome of this bacterium have been less successful because of the inability to differentiate true secreted substrates from those that are present due to lysis or sample preparation. We established and quantified secreted substrates from M. tuberculosis using a novel application of a known mass-spectrometric quantitative technique employing isobaric mass tags and applied it to culture supernatants from wild-type M. tuberculosis and mutants deficient for components of ESX-1. Using this technique, we sought to identify ESX-1 substrates and determine whether duplicate Cfp-10 paralogs are secreted in an ESX-1-dependent manner. This has enabled us to definitively illustrate that most of the Cfp-10 paralogs are secreted in the absence of functional ESX-1, uncovering the presence of alternative secretion systems that have not been characterized. Also, we paradoxically found a quantitative increase in the secretion of substrates dependent upon Sec in ESX-1 mutants, suggesting cross-talk between general and alternative secretion systems. Further characterization of these substrates by mass-spectrometric quantification of dominant-negative mutants gave further insight into the specificity and mechanism of ESX-1 substrate secretion.