Analysis of isoaspartate in peptides and proteins without the use of radioisotopes

A rapid and sensitive HPLC-based method for quantitating isoaspartate level s in peptides and proteins is described. The analyte is incubated for 40 mi n with S-adenosyl-L-methionine and the commercially available enzyme protei n L-isoaspartyl methyltransferase. Methylation of isoaspartyl sites results in stoichiometric production of S-adenosyl-L-homocysteine that is separate d from the other components of the reaction by reversed-phase HPLC and quan titated 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 int act proteins. Using a commercially available isoaspartyl peptide, the relat ionship between isoaspartate levels and S-adenosyl-L-homocysteine productio n was found to be linear and stoichiometric over a range of 5-250 pmol. Com pared to methods that measure [H-3]methanol production after methylation wi th S-adenosyl-L-[methyl-H-3]methionine, the HPLC method is safer, faster, l ess expensive, and equally sensitive. (C) 2000 Academic Press.