mNeuCode Empowers Targeted Proteome Analysis of Arginine Dimethylation

Characterization
of protein arginine dimethylation presents
significant
challenges due to its occurrence at the substoichiometric level. To
enable a targeted MS/MS analysis of these dimethylation sites, we
developed the mNeuCode (methyl-neutron-coding) tag by metabolically
labeling methylarginine with stable isotopes during cell culture,
which generated a diagnostic peak containing the NeuCode isotopologue
signature in a high-resolution MS scan. A software tool, termed NeuCodeFinder,
was developed for screening the NeuCode signatures in mass spectra.
Therefore, a targeted MS/MS workflow was established for proteome-wide
discovery of arginine dimethylation. The efficacy and utility were
demonstrated by identifying 176 arginine dimethylation sites residing
on 70 proteins in HeLa cells. Among them, 38% of the sites and 29%
of the dimethylated proteins are novel, including five novel arginine
dimethylation sites on the protein FAM98A, which is a substrate of
protein arginine methyltransferase 1 (PRMT1). Our results show that
deletion of FAM98A in HeLa cells suppressed cell migration, and importantly,
dimethylation-deficient mutation suppressed this process as well.
Therefore, the PRMT1-FAM98A pathway mediates cell migration possibly
through dimethylation of these newly identified sites of FAM98A. Our
study might drive the methodological shift from shotgun-based to targeted
proteome analysis for interrogation of the substoichiometric biomolecules
by using NeuCode-enabled techniques.