Improved Identification of Protease Cleavage Sites by In-gel Reductive Dimethylation.

Abstract

A critical step in the functional characterization of proteases is the identification of physiologically relevant substrates, which often starts with a collection of candidate proteins. To test these candidates and identify specific processing sites, in vitro cleavage assays are typically used, followed by polyacrylamide gel electrophoresis (SDS-PAGE) to separate and visualize the cleavage products. For the identification of cleavage sites, the sequences at the N- or C-terminal ends of the cleavage products need to be identified, which is the most challenging step in this procedure. Here, we describe a method for the reliable identification of the N-termini of polypeptides after separation by SDS-PAGE. The procedure relies on in-gel labeling of the N-terminal-free amino group by reductive dimethylation, followed by tryptic digestion and analysis of resulting peptides by mass spectrometry. N-terminal peptides are readily identified by the 28Da mass dimethyl tag linked to their first amino acid.