Adenosine dialdehyde (AdOx) is an indirect methyltransferase inhibitor broadly used in cell culture to accumulate methyl-accepting proteins in hypomethylated states for in vitro protein methylation analyses. In this study we included a translation inhibitor, cycloheximide, in the AdOx treatment of HeLa cells. The methyl-accepting proteins disappeared in the double treatment, indicating that they were most likely newly synthesized in the AdOx incubation period. AdOx treatment could also be used in combination with in vivo methylation, another technique frequently used to study protein methylation. AdOx treatment prior to in vivo methylation accumulated methyl-accepting proteins for the labeling reaction. The continued presence of AdOx in the in vivo labeling period decreased the methylation of the majority of in vivo methyl-accepting polypeptides. The level and pattern of the in vivo methylated polypeptides did not change after a 12-h chase, supporting the notion that the methylated polypeptide as well as the methyl groups on the modified polypeptides are stable. On the other hand, methylarginine-specific antibodies detected limited but consistent reduction of the methylarginine-containing proteins in AdOx-treated samples compared to the untreated ones. Thus, AdOx treatment probably only blocked a small fraction of stable protein methylation. Overall, it is likely that base-stable methylation are formed soon after the synthesis of the polypeptide and remain stable after the modification.
