High-performance liquid chromatographic method to measure protein L-isoaspartyl/D-aspartyl o-methyltransferase...

Protein L-isoaspartyl/D-aspartyl o-methyltransferase (PIMT) is a widely expressed protein repair enzyme that restores isomerized aspartyl residues to their normal configuration. Current methods for measuring PIMT activity have limited sensitivity or require radioactivity. We have developed a highly sensitive new assay method to measure PIMT activity in cell lysates. As a substrate, we used a fluorescently labeled delta sleep-inducing peptide (DSIP) that contains an isoaspartyl residue: 7-nitro-2,1,3-benzoxadiazole (NBD)-DSIP(isoAsp). The PIMT-catalyzed transfer of a methyl group onto this substrate can be detected with a simple high-performance liquid chromatography (HPLC) procedure. After the enzyme reaction, the methylated form of the peptide is stable and can be reproducibly separated from the unmethylated form in an acidic solvent and fluorometrically detected by HPLC. The limit of detection was estimated to be approximately 1 pmol of NBD-DSIP(isoAsp) (signal/noise ratio [S/N]=3), and the quantitation limit of the activity was approximately 18 microg of total cell lysate from HEK293 cells (10.7 pmol/min/mg protein). This assay method is sensitive enough to detect PIMT activity in biological samples without the use of radioisotopes, offering significant advantages over previously reported methods.