The full-length DNA encoding human PRMT5 was cloned into the pcDNA3.1 vector with an N-terminal Flag-tag, and the full-length DNA encoding MEP50 and PRMT1 was cloned into the pcDNA3.1 vector with a C terminal His6-tag individually. HEK293F cells were maintained in the FreeStyle 293 Expression Medium and transfected with PRMT5/MEP50 vectors or PRMT1 vector using FectoPRO according to Polyplus instructions. Cells were collected 48–72 h post-transfection, washed, and resuspended in a binding buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% glycerol, 20 mM imidazole, 1 mM tris(2-carboxyethyl)phosphine-HCl (TCEP), 0.1% Triton X-100, and protease inhibitors). The cells were lysed by sonication for 2 min and cleared by centrifugation at 15,000g for 10 min at 4 °C. The supernatant was subjected to protein purification through IMAC affinity chromatography (GE). The proteins were eluted using an elution buffer (50 mm Tris-HCl, pH 7.5, 300 mm NaCl, 10% glycerol, 250 mM imidazole, TCEP, 0.1% Triton X-100), and the eluents were analyzed by SDS-PAGE. Purified proteins were pooled, concentrated, and stored at −80 °C.

Protein arginine methylations are important post-translational modifications (PTMs) in eukaryotes, regulating many biological processes. However, traditional collision-based mass spectrometry methods inevitably cause neutral losses of methylarginines, preventing the deep mining of biologically important sites. Herein we developed an optimized mass spectrometry workflow based on electron-transfer dissociation (ETD) with supplemental activation for proteomic profiling of arginine methylation in human cells. Using symmetric dimethylarginine (sDMA) as an example, we show that the ETD-based optimized workflow significantly improved the identification and site localization of sDMA. Quantitative proteomics identified 138 novel sDMA sites as potential PRMT5 substrates in HeLa cells. Further biochemical studies on SERBP1, a newly identified PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric dimethylarginine in the central RGG/RG motif, and loss of either methylation caused increased the recruitment of SERBP1 to stress granules under oxidative stress. Overall, our optimized workflow not only enabled the identification and localization of extensive, nonoverlapping sDMA sites in human cells but also revealed novel PRMT5 substrates whose sDMA may play potentially important biological functions.