Although Mycobacterium avium ssp. paratuberculosis (M. ptb.) has long been recognised as the causative agent of Johne's disease (JD), there is a growing body of evidence that implicates this organism in Crohn's disease (CD) in humans. Although no causal link between the two has yet been proved, M. ptb. represents a pathogen of enormous economic significance to agriculture world-wide as well as one with potential risks to human health.Our laboratory is applying proteomic methodologies to elucidate not only the molecular mechanisms underlying M. ptb. pathogenesis in animals but also proteins that might represent biomarkers of paratuberculosis infection. Whilst the interaction between M. ptb. and host cells is poorly understood at the cellular level, it is reasonable to suppose that surface-exposed mycobacterial proteins are one of many factors that mediate infection and transmission. The thick, waxy external coating of mycobacteria, while imparting to these organisms their characteristic robustness, necessitates analysis strategies that maximise extraction of cellular proteins without compromising the integrity of the proteomic milieu. We have used the non-ionic detergent Triton X-114, which partitions into aqueous- and detergent-rich phases, to enrich hydrophobic proteins from cell-surface extracts of M. ptb. and the closely-related M. avium. Proteins were electrophoretically-separated, identified using mass spectrometry and subjected to bioinformatic analysis to determine their likely subcellular localisation. Thus far over three hundred proteins have been extracted from these two organisms and their usefulness as species-specific biomarkers or as a source of potential antigens is being evaluated through immunological screening.