Protease Substrate Identification Using N-terminomics.

Proteases are key regulators of vital biological processes, such as apoptosis, cell differentiation, viral infection and neurodegeneration. Proteases are tightly regulated, largely because proteolysis is a unique post-translational modification (PTM) that is essentially irreversible. In order to understand the role of proteases in health and disease, the identification of protease substrates is an important step toward our understanding of their biological functions. Classic approaches for the study of proteolysis in complex mixtures employ gel electrophoresis and mass spectrometry. Such approaches typically identify a few protein substrates at a time but often fail to identify specific cleavage site locations. In contrast, modern proteomic methods using enrichment of proteolytic protein fragments can lead to the identification of hundreds of modified peptides with precise cleavage site determination in a single experiment. In this manuscript, we will review recent advances in N-terminomics methods and highlight key studies that have taken advantage of these technologies to advance our understanding of the role of proteases in cellular physiology.