Heavy Methyl SILAC Metabolic Labeling of Human Cell Lines for High-Confidence Identification of R/K-Methylated Peptides by High-Resolution Mass Spectrometry

Abstract

Protein methylation is a widespread post-translational modification (PTM) involved in several important biological processes including, but not limited to, RNA splicing, signal transduction, translation, and DNA repair. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered today the most versatile and accurate technique to profile PTMs with high precision and proteome-wide depth; however, the identification of protein methylations by MS is still prone to high false discovery rates. In this chapter, we describe the heavy methyl SILAC metabolic labeling strategy that allows high-confidence identification of in vivo methyl-peptides by MS-based proteomics. We provide a general protocol that covers the steps of heavy methyl labeling of cultured cells, protein sample preparation, LC-MS/MS analysis, and downstream computational analysis of the acquired MS data.

Keywords: FDR; Heavy methyl SILAC; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Metabolic labeling; Protein methylation; Protein methyltransferases; Proteomics.