ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates

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.