title: Quantifying in vivo, site-specific changes in protein methylation with SILAC.
abstract: Interest in protein methylation has grown rapidly in recent years. Mass spectrometry-based proteomics is ideally suited to characterize protein modifications, but the multiplicity of methylated residues and the lack of efficient methods to enrich methylated proteins have limited the proteomic identification of protein methylation sites. In this protocol, we compare two metabolic labeling approaches, stable isotope labeling by amino acids in cell culture (SILAC) and its variant heavy methyl SILAC, for studying protein methylation. Instead of heavy lysine and arginine in the typical SILAC experiment, heavy methyl SILAC uses (13)C, (2)H methionine as the labeling amino acid. As cells convert methionine to S-adenosylmethionine, heavy methyl SILAC encodes a 4 Da mass tag for each methyl group, distinguishing between degrees of methylation is possible from mass difference alone. We provide a protocol for SILAC-based analyses of protein methylation and highlight the strengths and weaknesses of each method for targeted and proteomic analyses.
authors: Department of Pharmacology, University of Washington, 1959 NE Pacific Street, 357280, Seattle, WA, 98195, USA.
authors: Ho-Tak
authors: HT
authors: Lau
authors: Karen A
authors: KA
authors: Lewis
authors: Shao-En
authors: SE
authors: Ong
mesh_terms: D000478:Alkylation; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002853:Chromatography, Liquid; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D008745:Methylation; D051379:Mice; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D000596:Amino Acids; D004220:Disulfides; D010455:Peptides; D011506:Proteins