SIMPATICO-Streamlined Isolation of the N-Terminome via Phosphonate Tagging and Coordination-Based Depletion

Protein processing is critical for regulating cellular functions. Therefore, there is considerable interest in developing effective proteome-wide strategies to identify protease cleavage products to enhance our understanding of proteolytic pathways and their perturbation in diseases. Here, we present a streamlined N-termini proteome analysis leveraging N-Hydroxysuccinimide (NHS) ester chemistry. At the protein level, N-terminal amines (naturally occurring protein N-termini, lysines, or protease-generated N-termini) are blocked directly in the cell lysate, and tryptic digestion is performed straight after, without the need for any buffer exchange. The internal tryptic peptides are tagged with a phosphonate moiety and subsequently depleted via metal-based coordination, keeping exclusively N-blocked terminal peptides for analysis by mass spectrometry. We demonstrate the applicability of this approach by monitoring proteolytic events induced by intrinsic apoptotic pathway. Our approach identified more than 690 aspartyl-cleaved proteins in response to Staurosporine-induced apoptosis, including many previously unknown substrates and cleavage sites. The presented approach is, therefore, a straightforward and robust method for mass spectrometry-based identification of caspase-generated cleavage products. Although demonstrated here in the context of caspase-mediated proteolysis, the underlying workflow is not restricted to this protease class and should be broadly applicable to the study of other proteolytic events.