title: Analysis of isoaspartate in peptides and proteins without the use of radioisotopes.
abstract: A rapid and sensitive HPLC-based method for quantitating isoaspartate levels in peptides and proteins is described. The analyte is incubated for 40 min with S-adenosyl-l-methionine and the commercially available enzyme protein l-isoaspartyl methyltransferase. Methylation of isoaspartyl sites results in stoichiometric production of S-adenosyl-l-homocysteine that is separated from the other components of the reaction by reversed-phase HPLC and quantitated online by absorbance at 260 nm. This method can accurately detect 5 pmol or less of isoaspartate and works with tryptic digests as well as intact proteins. Using a commercially available isoaspartyl peptide, the relationship between isoaspartate levels and S-adenosyl-l-homocysteine production was found to be linear and stoichiometric over a range of 5-250 pmol. Compared to methods that measure [(3)H]methanol production after methylation with S-adenosyl-l-[methyl-(3)H]methionine, the HPLC method is safer, faster, less expensive, and equally sensitive.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA.
authors.forename: B T
authors.initials: BT
authors.lastname: Schurter
authors.forename: D W
authors.initials: DW
authors.lastname: Aswad
mesh_terms: D001224:Aspartic Acid / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003701:Delta Sleep-Inducing Peptide / Q000032:analysis; D008745:Methylation; D010455:Peptides / Q000737:chemistry*; D011496:Protein Methyltransferases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012435:S-Adenosylhomocysteine / Q000032:analysis; D012436:S-Adenosylmethionine / Q000737:chemistry; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D010455:Peptides; D011506:Proteins; D001224:Aspartic Acid; D003701:Delta Sleep-Inducing Peptide; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D011496:Protein Methyltransferases