Large-Scale Identification of the Arginine Methylome by Mass Spectrometry.

Autor: Sylvestersen KB; Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark., Nielsen ML; Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark.
Jazyk: angličtina
Zdroj: Current protocols in protein science [Curr Protoc Protein Sci] 2015 Nov 02; Vol. 82, pp. 24.7.1-24.7.17. Date of Electronic Publication: 2015 Nov 02.
DOI: 10.1002/0471140864.ps2407s82
Abstrakt: 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.
(Copyright © 2015 John Wiley & Sons, Inc.)
Databáze: MEDLINE
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