Decoding Arginine Dimethylation Isomers via pH-Tuned Reactivity with Methylglyoxal: A Chemical Approach for Functional Proteomics

Arginine dimethylation, encompassing asymmetric and symmetric configurations, represents a fundamental post-translational modification. Despite sharing identical chemical formulas, the two arginine dimethylation isomers exhibit different or even opposite biological effects. Therefore, it is necessary to determine their specific structure before conducting a further biological investigation. However, current methods for arginine dimethylation analysis face great challenges in efficient isomer differentiation, preventing the functional investigation of arginine dimethylation. To overcome this obstacle, herein, we introduce a novel chemical strategy leveraging pH-tuned reactivity with methylglyoxal (MGO) to decode these dimethylation isomers. By utilizing molecular dynamics simulation analysis, we revealed the different chemical reactivities of asymmetrically and symmetrically dimethylated arginine when reacted with MGO at different pH conditions. This property enabled the development of a pH-tuned chemical strategy by combining the MGO reaction with boronate affinity enrichment to simultaneously enrich and differentiate the dimethylation isomers. This strategy can effectively distinguish dimethylated arginine isomers in complex cell samples, and the good feasibility of this strategy was verified by orthogonal validation with the neutral loss. Of the obtained data set, this strategy identified sDMA at R112 of SNRPN, which is confirmed to be modified by PRMT5. Further functional analysis reveals its crucial role in maintaining protein stability and in regulating spliceosome assembly. Overall, by transforming the inherent pH sensitivity of MGO reactions into a powerful analytical tool, our work establishes the first chemical platform for functional proteomic dissection of arginine dimethylation isomers, which paves the way for further regulating mechanism investigations of protein methylation.