Large‐Scale Identification of the Arginine Methylome by Mass Spectrometry

Abstract

The attachment of one or more methylation groups to the side chain of arginine residues is a regulatory mechanism for cellular proteins. Recent advances in mass spectrometry-based characterization allow comprehensive identification of arginine methylation sites by peptide-level enrichment strategies. Described in this unit is a 4-day protocol for enrichment of arginine-methylated peptides and subsequent identification of thousands of distinct sites by mass spectrometry. Specifically, the protocol explains step-by-step sample preparation, enrichment using commercially available antibodies, prefractionation using strong cation exchange, and identification using liquid chromatography coupled to tandem mass spectrometry. A strategy for relative quantification is described using stable isotope labeling by amino acids in cell culture (SILAC). Approaches for analysis of arginine methylation site occupancy are also discussed. Collectively, the unit describes the essential parameters required for a successful and comprehensive experiment detailing the arginine methylome.

Separation of Isobaric Mono- and Dimethylated RGG-Repeat Peptides by Differential Ion Mobility-Mass Spectrometry

“…Chymotrypsin also circumvents this limitation of trypsin. 35 ESI-MS of solutions containing each selected peptide in 50/ 50% (v/v) water/acetonitrile resulted in the formation of each of the six peptide ions in the triply and quadruply protonated charge states in relatively high abundances under these conditions (SI Figure S2). Thus, the FAIMS-MS spectra of the 3+ and 4+ charged states are reported here (see below).…”

“…Moreover, trypsin does not cleave as efficiently at methylated arginine residues, which can preclude the relative quantification of methylated peptides in respect to the corresponding unmethylated peptides with the same length and sequence. Chymotrypsin also circumvents this limitation of trypsin …”