Protein methylation characterization using NMR without isotopic labeling

Protein methylation is crucial in epigenetics, and targeting the involved methyltransferases shows great potential for therapeutic intervention with several inhibitors in clinical trials for oncology indications. Therefore, char-acterization of protein methylation is essential for understanding the methyltransferase function and discovering chemical inhibitors and antagonists. While NMR has been used to measure methylation rates, isotopic labeling of protein or methyl donors can be costly and cannot characterize demethylation of proteins extracted from natural sources. Our method employs a four-quantum filter 1H -13C experiment that selectively detects methyl groups, providing a simple way to characterize methylation and demethylation features of methyltransferases and demethylases, respectively, without requiring isotopic labeling. In our experiments, we successfully observed the methylation of H3 under lysate from various cells and tissues of mice with cancerous growth. The results revealed that H3 undergoes both mono-and dimethylation in all the tested lysates, but at varying rates and degrees. Significantly lower H3 methylation rates and levels were observed in both cervical tumor and breast tumor lysates compared with the corresponding cancerous cells and healthy cells lysates. These findings high -light the variability of histone H3 methylation patterns among healthy cells, cancerous cells, tumor tissues, and different tumor types, and suggest that this method has great potential in facilitating the development of effective interventions against these diseases. By characterizing the methylation features of suspected tumors or areas of concern, it provides valuable insights into the underlying mechanisms of cancer development and aids in identifying potential targets for therapeutic interventions.