Propargylic <i>Se</i>-adenosyl-<scp>l</scp>-selenomethionine: A Chemical Tool for Methylome Analysis
Yoshihiro Sohtome, Tadahiro Shimazu, Yoichi Shinkai, Mikiko Sodeoka
Abstract
-adenosyl-l-selenomethionine (ProSeAM, also called SeAdoYn). Specifically, we draw heavily from quantitative proteomics that yields information about the differences between two samples utilizing LC-MS/MS analysis. By exploiting the use of ProSeAM, we have prepared the requisite two samples for quantitative methylome analysis. The structural difference between ProSeAM and the parent SAM is so small that the quantity of modification of the protein substrate with this artificial cofactor reflects, to a large extent, levels of activity of the MTase of interest with SAM. First, we identified that the addition of exogenous recombinant MTase (methylation accel), a natural catalyst, enhances the generation of the corresponding propargylated product even in the cell lysate. Then, we applied the principle to isotope label-free quantification with HEK293T cell lysates. By comparing the intensity of LC-MS/MS signals in the absence and presence of the MTase, we have successfully correlated the MTase substrates. We have currently applied the concept to the stable isotope label-based quantification, SILAC (stable isotope labeling by amino acids in cell culture). The strategy merging ProSeAM/MTase/SILAC (PMS) is uniquely versatile and programmable. We can choose suitable cell lines, subcellular fractions (i.e.; whole lysate or mitochondria), and genotypes as required. In particular, we would like to emphasize that the use of cell lysates derived from disease-associated MTase knockouts (KOs) holds vast potential to discover functionally unknown but biologically important methylation events. By adding ProSeAM and a recombinant MTase to the lysates derived from KO cells, we successfully characterized unprecedented nonhistone substrates of several MTases. Furthermore, this chemoproteomic procedure can be applied to explore MTase inhibitors (methylation brake). The combined strategy with ProSeAM/inhibitor/SILAC (PIS) offers intriguing opportunities to explore nonhistone methylation inhibitors.Considering that SAM is the second most widely used enzyme-substrate following ATP, the interdisciplinary research between chemistry and biology using SAM analogs has a potentially huge impact on a wide range of research fields associated with biological methylation. We hope that this Account will help to further delineate the biological function of this important class of enzymatic reaction.