(307d) Improved Discrimination of Asymmetric and Symmetric Arginine Dimethylation By Optimization of the Normalized Collision Energy in LC-MS Proteomics

Protein arginine methylation regulates diverse biological processes including signaling, metabolism, splicing, and transcription. Despite its important biological roles, arginine dimethylation remains an understudied post-translational modification. Partly, this is because the two forms of arginine dimethylation, asymmetric and symmetric dimethyl arginine, are isobaric and therefore indistinguishable by traditional mass spectrometry techniques. Thus, there exists a need for methods that can differentiate these two modifications. Here, we report that increasing the normalized collision energy (NCE) in a higher-energy collisional dissociation (HCD) cell of a Q-Exactive Plus mass spectrometer increases the generation of characteristic neutral losses (NL) that can distinguish asymmetric and symmetric dimethyl arginine. By analyzing both synthetic and endogenous dimethyl-peptides, we identify an optimal NCE value that maximizes NL generation and also improves methyl-peptide identification. Using two orthogonal methyl peptide enrichment strategies, high pH strong cation exchange (SCX) and immunoaffinity purification (IAP), we demonstrate that the optimal NCE increases improves NL-based ADMA and SDMA annotation and dimethyl peptide identifications by 125% and 17%, respectively, compared to the standard NCE. This simple parameter change will greatly facilitate the identification and annotation of asymmetric and symmetric dimethyl arginine in mass spectrometry-based methyl-proteomics to improve our understanding of how these modifications differentially regulate protein function.