Activity-Based Protein Profiling of Bacterial Monooxygenases

Abstract

Activity-based protein profiling (ABPP) is a proteomics approach that enables the selective molecular characterization of specific proteins within complex mixtures. A variety of activity-based approaches have been used to characterize some important bacterial monooxygenases such as ammonia monooxygenase (AMO) that have resisted purification-based characterization. In this report, we describe a 2-step ABPP method for AMO and several other types of bacterial monooxygenases that play key roles in biogeochemical cycles and processes. The focus of this report is on SDS-PAGE-based methods to detect fluorescently labeled monooxygenase components. This report also draws attention to various features of these types of analyses that can impact the use of ABPP approaches for the quantification of monooxygenase components and their detection in different compartments of bacterial cells.

Keywords: Activity-based protein profiling; Diyne probe; Monooxygenases; Near-infrared (NIR) fluorescent imaging; SDS-PAGE.