We report here an isotopic labeling and mass spectrometric method to rapidly identify S-adenosylmethionine (AdoMet)-dependent methylation products. In the presence of CH(3)- and CD(3)-labeled AdoMet, a methyl transfer product appears as a doublet separated by 3 Da in a mass spectrum, while other compounds show their normal isotopic distribution. Based on this unique isotopic pattern, methylation product(s) can be easily detected even from a mixture of cellular components. To validate our method, the product of human thiopurine methyltransferase (TPMT, EC 2.1.1.67) has been successfully identified from both an in vitro assay and a whole-cell assay. This method is generally applicable to AdoMet-dependent transmethylation and other group-transfer reactions, and constitutes the first example of a general strategy of enzyme-transferred isotope patterns (ETIPs) analysis.
Deuterium Escherichia coli