Activity-based lipid esterase profi ling of m. Bovis BCG at different metabolic states using tetrahydrolipstatin (THL) as bait

Abstract

© Springer Science+Business Media New York 2017. This chapter provides a step-by-step protocol using activity-based protein profiling (ABPP) as a chemicalproteomic tool to survey the antibiotic properties of a small molecule. Here, we investigate the molecular mechanism behind the bactericidal activity of tetrahydrolipstatin (THL). ABPP relies on small molecule probes that target the active site of specific enzymes in complex proteomes. These probes in turn are equipped with a reporter tag that allows capturing, visualization, enrichment, identifi cation, and quantify cation of its targets either in vitro or in situ. THL possesses bactericidal activities, but its precise spectrum of molecular targets is poorly characterized. Here, we used THL analogs functionalized to enable Huisgen-base cycloaddition, commonly known as “click chemistry,” to identify target proteins after enrichment from mycobacteria cell lysates obtained from different physiological conditions.