Proteolytic cleavage is a ubiquitous post-translational modification of proteins, responsible for protein signalling, activation, localisation and ultimately degradation. Due to a variety of experimental limitations this important physiological process has been largely understudied, particularly in prokaryotes and archaea. Proteases and proteolytic processing have been linked to virulence in a variety of infectious diseases. Therefore experimental investigation of proteolytic cleavage is needed to determine the extent and functional roles of protein processing in the production of mature proteins.

The primary focus of this study is to sequence the N-terminal sequences of mature protein products of prokaryotic pathogens, and to characterise the resulting mature protein fragments. N-terminal sequence data can be used to identify true protein start sites and any downstream post-translational processing. The genome-reduced agriculturally-important pathogen Mycoplasma hyopneumoniae, was selected as a model organism for these studies. There is a large body of literature demonstrating extensive proteolytic processing of many highly expressed adhesins which are critical for pathogenesis.

Using dimethyl labelling of protein amine groups, including native N-termini, the data obtained from mass spectrometry analysis has provided evidence confirming the start sites of protein translation and complementary data pinpointing the exact sites of proteolytic cleavage. In addition, the data indicates that an unprecedented number of cytosolic proteins are targets for post-translational processing on the cell surface of pathogenic bacteria. Many of these proteins have been described as “moonlighting” proteins in the literature and have been implicated in pathogenesis in other organisms.