Emerging Technologies to Map the Protein Methylome

Abstract

Protein methylation plays an integral role in cellular signaling, most notably by modulating proteins bound at chromatin, and increasingly through regulation of non-histone proteins. One central challenge in understanding how methylation acts in signaling is identifying and measuring protein methylation. This includes locus-specific modification of histones, on individual non-histone proteins, and globally across the proteome. Protein methylation has traditionally been studied using candidate approaches such as methylation-specific antibodies, mapping of post-translational modifications by mass spectrometry, and radioactive labeling to characterize methylation on target proteins. Recent developments have provided new approaches to identify methylated proteins, measure methylation levels, identify substrates of methyltransferase enzymes, and match methylated proteins to methyl-specific reader domains. Methyl-binding protein domains and improved antibodies with broad specificity for methylated proteins are being used to characterize the “protein methylome”. They also have the potential to be used in high-throughput assays for inhibitor screens and drug development. These tools are often coupled to improvements in mass spectrometry to quickly identify methylated residues, and to protein microarrays, where they can be used to screen for methylated proteins. Finally, new chemical biology strategies are being used to probe the function of methyltransferases, demethylases and methyl-binding “reader” domains. These tools are creating a “system-level” understanding of protein methylation, and integrating protein methylation into broader signaling processes.

Set7/9, a methyltransferase, regulates the thermogenic program during brown adipocyte differentiation through the modulation of p53 acetylation

“…According to the accumulated evidences that lysine methylation is not histone-specific, lysine methyltransferases (KMTs) and lysine demethylases (KDMs) are now widely accepted. A recent massive proteomic analysis enabled mining of methylated proteins from whole cell extracts or enriched fractions (Carlson and Gozani, 2014;Chick et al, 2015;Wang et al, 2015). Only a few studies have been performed with regard to the relatedness of PTMs during brown adipocyte differentiation.…”

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme

“…Only a few of these sites have been associated with specific KMTs, so a large number of enzyme/substrate relationships remain to be discovered (14). Techniques for linking KMTs to non-histone substrates are limited, and this remains a major challenge in understanding how lysine methylation participates in wider signaling processes (15). We recently reported a proteomic strategy to identify and measure lysine methylation by using the three malignant brain tumor domain repeats (3xMBT) of L3MBTL1 as a universal affinity reagent to capture modified proteins (10,16).…”

Update: Post-translational protein modifications in plant metabolism

“…Recent studies with nonplant material (e.g. mammalian cells and yeast [Saccharomyces cerevisiae]) using various novel enrichment techniques followed by MS/MS showed that high numbers of Arg (monomethylation and demethylation) and Lys (monodimethylation and trimethylation) are methylated (for an excellent overview on innovative methodologies, see Carlson and Gozani [2014]). These methylations are carried out by Arg or Lys methyltransferases (PRMTs and PKMTs, respectively) with S-adenosylmethionine (AdoMet) as methyl donor, thereby releasing S-adenosyl-homo-Cys (Biggar and Li, 2015) and coupling the cellular metabolic state to this PTM.…”