How to Detect Protein Methylation Using Proteomics

Protein methylation is a ubiquitous and critical post-translational modification (PTM) in eukaryotes that occurs primarily on lysine and arginine residues. It is not to be confused with DNA and RNA methylation, although both protein and DNA/RNA methylation have profound effects on cellular epigenetics. 

Methylation of arginine is known to be involved in regulating gene transcription, RNA metabolism, DNA damage repair, and signal transduction. Known to form three isotypes, methylated arginine is commonly found in nuclear and cytoplasmic proteins that shuttle between the two compartments, as well as sites on histone tails.¹ Because of its crucial role in various cellular processes, it’s perhaps not surprising that cancer cells are particularly susceptible to interventions that target the inhibition of arginine methylation. 

So, how can proteomics be used to detect protein methylation in general, and, importantly, how can it help to distinguish between different isotypes of methylated arginine?

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Assays to Detect Protein Methylation

PTMScan^® Methylome Proteomics is a novel and groundbreaking technology developed by my colleagues at CST that leverages patented PTMScan assay technology to enable scientists to enrich for methylated peptides (Figure 1) for subsequent liquid chromatography-mass spectrometry (LC-MS) profiling.² 

The PTMScan methylation-based kits specifically enrich for the following methylated arginine and methylated lysine residues:

• Methylarginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA)
• Methyllysine, dimethyllysine, and trimethyllysine, which are also collectively referred to as pan-methyllysine. 

Figure 1. Antibodies specific to methylated arginine and lysine for use in PTMScan Methylome Proteomics assay kits.

When PTMScan Methylome Proteomics first debuted in 2014, we used the technology to identify over 1,000 sites of arginine methylation and hundreds of lysine-methylated sites in human and mouse cell lines.

The research, published in the Molecular & Cellular Proteomics paper Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation by Guo et al. not only confirmed many previously identified forms of protein methylation, it also more than doubled the total number of previously identified protein methylation sites.² 

Interestingly, some of these sites were found on known methyltransferases like EZH1, EZH2, and SETDB1, which may automethylate themselves. This result suggests that the activity of certain methylating enzymes could be modulated through a feedback loop mechanism; a finding that may have broad epigenetic implications.³

Distinguishing Symmetric (SDMA) vs Asymmetric Dimethylarginine (ADMA)

The defining advantage of our dimethylarginine antibodies is their ability to differentiate between the symmetric (SDMA) and asymmetric forms (ADMA) of methylated arginine, which are identical in mass and, therefore, are difficult to distinguish using traditional mass spectrometry techniques.

Why is distinguishing between SDMA and ADMA important? This ability can aid in cancer research. 

Studies from multiple laboratories employing PTMScan Methylome Proteomics have identified RNA splicing factors as crucial targets of protein arginine methyltransferases (PRMTs).^4,5,6 PRMTs are a nine-member family of proteins that add methyl groups to arginine residues to produce both singly and doubly methylated arginine, including symmetric and asymmetric dimethylated arginine (Figure 2). PRMTs are often overexpressed in many types of cancer, and PRMT5 is the primary arginine methyltransferase responsible for the formation of SDMA.^1,4,5

Figure 2. PRMTs catalyze the addition of methyl groups to arginine. 

Some of the proteins that are symmetrically dimethylated by PRMT5 are members of the RNA splicesome, and inhibition of PRMT5 has been shown to lead to the loss of key proteins involved in oncogenesis.⁷ Cancer cells often have elevated levels of PRMT5, and therefore, because PRMT5 activity correlates with SDMA levels, the ability to detect the symmetric form of methylated arginine is important in cancer research.

In addition to the arginine methylation kits described above, the PTMScan^® Pan-Methyl Lysine Kit #14809 and PTMScan^® HS Pan-Methyl Lysine Kit #28411 provide a balanced enrichment for all three forms of lysine methylation: mono-, di-, and trimethyllysine. The PTMScan^® Mono-Methyl Lysine Motif Kit #16892, in contrast, provides an improved enrichment specifically for the monomethyllysine form. While the abundance of methylated lysine sites is considerably lower than arginine methylation, dysregulation of lysine methylation is associated with neurological and developmental disorders, as well as cancer.

Contributions of PTMScan to Proteomics Research 

Research in laboratories worldwide employing PTMScan Methylome technology have uncovered novel sites of lysine and arginine methylation proteins, helping to elucidate the mechanisms of oncogenesis in a variety of cell types.^5,8

To explore identified lysine and arginine methylation sites in the literature, please visit PhosphoSitePlus our free, online database of PTMs, powered by CST.

Importantly, the PTMScan Methylome technology, combined with quantitative LC-MS/MS, can provide researchers with the sites of methylation and magnitude of change for hundreds to thousands of sites in a series of experiments.

PTMScan Methylome antibodies have been instrumental tools for scientists, helping to reveal the changes in protein methylation occurring in cell lines and tissues, particularly those in melanoma, lymphoma, and pancreatic cancers.

PTMScan Antibodies for Methylation Proteomics

*The pilot kits listed above provide enough immunoaffinity beads and IAP buffer for three enrichments, while the full kits provide enough for ten enrichments.

The product list above includes PTMScan HS kits, where the HS designates that the antibodies are coupled to magnetic beads. This simplifies the workflow at the lab bench, and importantly, these kits require less sample and are amenable to automation.

Select References:    

1. Bedford MT, Richard S. Arginine methylation an emerging regulator of protein function. Mol Cell. 2005;18(3):263-272. doi:10.1016/j.molcel.2005.04.003
2. Guo A, Gu H, Zhou J, et al. Immunoaffinity enrichment and mass spectrometry analysis of protein methylation. Mol Cell Proteomics. 2014;13(1):372-387. doi:10.1074/mcp.O113.027870
3. Lee CH, Yu JR, Granat J, et al. Automethylation of PRC2 promotes H3K27 methylation and is impaired in H3K27M pediatric glioma. Genes Dev. 2019;33(19-20):1428-1440. doi:10.1101/gad.328773.119
4. AbuHammad S, Cullinane C, Martin C, et al. Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma [published correction appears in Proc Natl Acad Sci U S A. 2020 Apr 28;117(17):9644-9645]. Proc Natl Acad Sci U S A. 2019;116(36):17990-18000. doi:10.1073/pnas.1901323116
5. Musiani D, Bok J, Massignani E, et al. Proteomics profiling of arginine methylation defines PRMT5 substrate specificity. Sci Signal. 2019;12(575):eaat8388. Published 2019 Apr 2. doi:10.1126/scisignal.aat8388
6. Radzisheuskaya A, Shliaha PV, Grinev V, et al. PRMT5 methylome profiling uncovers a direct link to splicing regulation in acute myeloid leukemia. Nat Struct Mol Biol. 2019;26(11):999-1012. doi:10.1038/s41594-019-0313-z
7. Li, Wj., He, Yh., Yang, Jj. et al. Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth. Nat Commun 12, 1946 (2021).
8. Hartel NG, Chew B, Qin J, Xu J, Graham NA. Deep Protein Methylation Profiling by Combined Chemical and Immunoaffinity Approaches Reveals Novel PRMT1 Targets. Mol Cell Proteomics. 2019;18(11):2149-2164. doi:10.1074/mcp.RA119.001625

Alexandra Foley, Scientific Marketing Writer and CST Blog Manager, contributed to writing this post.23-BPA-72850