"Shaving" Live Bacterial Cells with Proteases for Proteomic Analysis of Surface Proteins.

Surface proteins are essential molecules for the interplay between cells and the environment. They participate in many biological processes including transport, adhesion, cell-cell recognition, signaling, and other cell interactions. In pathogenic microorganisms, these molecules may act as virulence or cytotoxicity factors. Analyzing the set of surface proteins is critical to understand these processes and to identify possible targets that can be the starting point for other studies or discoveries (e.g., vaccines or diagnostics). Here I describe a proteomic procedure to identify in a fast and reliable way a set of surface-exposed proteins in bacteria, the methodology of which can be adapted to other biological systems (unicellular fungi, parasites). The protocol presented here involves "shaving" the cells cultured in broth with proteases followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and analysis of the generated peptides. This method overcomes some important limitations of the first-generation, gel based proteomics techniques, and the "shaving" approach allows one to identify which domains from identified proteins are more accessible to proteases. These identified proteins have the highest potential to be recognized by antibodies, and thus permits the identification of potential epitopes or antigens.