Protein processing characterized by a gel-free proteomics approach.

We describe a method for the specific isolation of representative N-terminal peptides of proteins and their proteolytic fragments. Their isolation is based on a gel-free, peptide-centric proteomics approach using the principle of diagonal chromatography. We will indicate that the introduction of an altered chemical property to internal peptides holding a free alpha-N-terminus results in altered column retention of these peptides, thereby enabling the isolation and further characterization by mass spectrometry of N-terminal peptides. Besides pointing to changes in protein expression levels when performing such proteome surveys in a differential modus, protease specificity and substrate repertoires can be allocated since both are specified by neo-N-termini generated after a protease cleavage event. As such, our gel-free proteomics technology is widely applicable and amenable for a variety of proteome-driven protease degradomics research.