Herein, we introduced ortho-phthalaldehyde (OPA) for blocking free amino groups and established a simple and robust method for comprehensive profiling of protein terminome based on strong cation exchange chromatography (SCX) fractionation. With the highly efficient and chemoseletive amine-group blocking, we identified 2271 canonical human protein N-termini, 1650 canonical human protein C-termini, as well as 645 protein neo-N-termini from HeLa cells.

Detection of (neo-)N-terminal peptides is essential for identifying protease cleavage sites . We here present an update of a well-established and efficient selection method for enriching N-terminal peptides out of peptide mixtures: N-terminal COFRADIC (COmbined FRActional DIagonal Chromatography). This method is based on the old concept of diagonal chromatography, which involves a peptide modification step in between otherwise identical chromatographic separations, with this modification step finally allowing for the isolation of N-terminal peptides by longer retention of non-N-terminal peptides on the resin. N-terminal COFRADIC has been successfully applied in many protease-centric studies, as well as for studies on protein alpha-N-acetylation and on characterizing alternative translation initiation events.