Analysis of Isoaspartate in Peptides and Proteins without the Use of Radioisotopes

✍ Scribed by Brandon T. Schurter; Dana W. Aswad

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 65 KB
• Volume: 282
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4601

✦ Synopsis

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.