Improved discrimination of asymmetric and symmetric protein arginine dimethylation by optimizing the normalized collision energy in LC-MS proteomics

Here we optimize the HCD Normalized Collision Energy (NCE) in order to generate neutral losses of monomethylamine and dimethylamine which can be lost by symmetric dimethylarginine and asymmetric dimethylarginine, respectively. By testing a range of NCEs across two different methyl enrichment techniques, we identified the optimal NCE as 32 to both generate neutral losses and to improve identifications of dimethyl PSMs. We achieve a 2-to-3 fold increase in the number of unique DMA peptides assigned neutral losses desginating them as ADMA or SDMA. This method will be useful to others interested in analyzing dimethylarginine peptides through mass spectrometry, where ADMA and SDMA have identical masses but differing neutral losses, and differing biological functions.