Characterisation of proteolytic process by in-gel protein N-terminal derivatisation using MS-based approach in Drosophila

摘要

Herein, we developed a new strategy adapted to follow proteolytic processes in order to provide insight into the biological mechanism of innate immunity. This approach allowed detecting one specific cleavage site after proteolytic maturation of Persephone incubated with pathogen virulence factors. Thanks to our labelling in gel method, we demonstrated that Persephone activation required pathogen proteolytic activities but with a differential mechanism according to the nature of pathogen protease. The cleavage observed in this highly proteolytic sensitive region could constitute the first step of a sequential activation of Psh. The increased ionization efficiency afforded by the TMPP tag enhances the detection of low abundance peptides and should therefore allow the in vivo study of proteolytic processes occurring in Drosophila blood. As this method adds only one step to the classical in-gel proteomic analysis, it can be easily implemented for the characterisation of proteolytic products.