Patent ID: 8367366
Filing Date: 2013-02-05
Classification: C12Q,G01N

Abstract:
1. A method for characterizing the activity of an enzyme or enzyme mixture comprising a methylase or demethylase, the method comprising: (a) incubating the enzyme or enzyme mixture in vitro with a polypeptide substrate under conditions that allow methylation and/or demethylation of the polypeptide substrate to add methyl groups to or remove methyl groups from one or more lysine residues of the polypeptide substrate to generate an enzymatically methylated and/or demethylated polypeptide, wherein the polypeptide substrate is a peptide fragment of a full-length protein; (b) reacting the enzymatically methylated and/or demethylated polypeptide with one or more substances to non-enzymatically react with the polypeptide to add one or more methyl groups to the polypeptide to generate a fully dimethylated or trimethylated polypeptide that is fully dimethylated or trimethylated at least one lysine residue; wherein the methyl groups non-enzymatically added to the polypeptide differ in molecular weight from the added or removed methyl groups of the enzymatically methylated and/or demethylated protein or peptide; and (c) subjecting the fully methylated polypeptide to mass spectrometry to quantitatively determine a ratio of enzymatically produced nonmethyl, monomethyl, and dimethyl and optionally trimethyl lysine species at least one lysine residue.