Methyl-Specific Capture Reagents

Naturally occurring methyl-lysine-binding domains provide alternative reagents to antibodies for the enrichment of methylated peptides or proteins. This approach is based on the observation that some methyl-lysine-binding domains, such as the triple MBT domains [3xMBT] of the protein L3MBTL1 bind to mono- and dimethylated lysine [105]. The 3xMBT domain exhibits minimal sequence specificity and thus has potential for pan methyl-lysine capture. A protocol for lysine methylome analysis has been developed using a recombinant 3xMBT-glutathione 5-transferase (GST) anchored to beads functionalized with reduced glutathione [106]. Proteins enriched by 3xMBT can be separated by SDS-PAGE and analyzed by either western blotting or ingel digestion with trypsin and LC-MS/MS. In this technique, a binding-null, inactive point mutant (D355N) of 3xMBT provides a negative control. SILAC labeling enables quantitative comparison of proteins captured by native 3xMBT and D355N mutant to discriminate specific from nonspecific binding and aid identifications by LC-MS/MS [106].

Immobilization of novel heterochromatin protein 1 p-chromodomain also provides a bait to capture methylated proteins, identifying dimethyl-lysine and lysine trimethylation sites [107]. It is interesting to note that there is no overlap among the methylation sites identified by this study and the 3xMBT domain capture study, which may be related to the use of different proteases prior to enrichment: trypsin [106] and Arg-C, Glu-C, chymotrypsin, and elastase [107].

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