SIMPATICO is a streamlined N-termini proteome workflow which leverages 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 LC-MS/MS.