D.B.L. is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (a.k.a. EMD in Canada and the US), Pfizer, and Takeda.

<ol>
	<li>Bedford, M. T., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methylation+in+mammals:+who,+what,+and+why.">Protein arginine methylation in mammals: who, what, and why.</a> Molecular Cell. 33 (1), 1-13 (2009).</li><li>Blanc, R. S., Richard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation:+The+coming+of+age.">Arginine methylation: The coming of age.</a> Molecular Cell. 65 (1), 8-24 (2017).</li><li>Wu, Q., Schapira, M., Arrowsmith, C. H., Barsyte-Lovejoy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methylation:+from+enigmatic+functions+to+therapeutic+targeting.">Protein arginine methylation: from enigmatic functions to therapeutic targeting.</a> Nature Reviews. Drug Discovery. 20 (7), 509-530 (2021).</li><li>Hutchinson, A., Seitova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+PRMT+proteins+using+the+baculovirus+expression+vector+system.">Production of recombinant PRMT proteins using the baculovirus expression vector system.</a> Journal of Visualized Experiments. (173), e62510 (2021).</li><li>Dhar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+major+Type+I+arginine+methyltransferase+PRMT1+causes+substrate+scavenging+by+other+PRMTs.">Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs.</a> Scientific Reports. 3, 1311 (2013).</li><li>Maniaci, M., Marini, F., Massignani, E., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mass+spectrometry-based+proteomics+approach+for+global+and+high-confidence+protein+R-methylation+analysis.">A mass spectrometry-based proteomics approach for global and high-confidence protein R-methylation analysis.</a> Journal of Visualized Experiments. (182), e62409 (2022).</li><li>Thandapani, P., O'Connor, T. R., Bailey, T. L., Richard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+RGG/RG+motif.">Defining the RGG/RG motif.</a> Molecular Cell. 50 (5), 613-623 (2013).</li><li>Habisch, H., Zhang, F., Zhou, Q., Madl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+arginine+methylome+by+nuclear+magnetic+resonance+spectroscopy.">Exploring the arginine methylome by nuclear magnetic resonance spectroscopy.</a> Journal of Visualized Experiments. (178), e63245 (2021).</li><li>Szewczyk, M. M., Vu, V., Barsyte-Lovejoy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+methods+to+study+protein+arginine+methyltransferase+1-9+activity+in+cells.">Quantitative methods to study protein arginine methyltransferase 1-9 activity in cells.</a> Journal of Visualized Experiments. (174), e62418 (2021).</li><li>Poulard, C., Jacquemetton, J., Valantin, J., Treilleux, I., Le Romancer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+ligation+assay+allows+the+detection,+localization,+and+quantification+of+protein+arginine+methylation+in+fixed+tissue.">Proximity ligation assay allows the detection, localization, and quantification of protein arginine methylation in fixed tissue.</a> Journal of Visualized Experiments. (185), e64294 (2022).</li></ol>

The authors have nothing to disclose.

Protein arginine methylation is a posttranslational modification that is involved in the regulation of a wide spectrum of biological processes1,2,3. The nine members of the protein arginine methyltransferases (PRMTs) family can methylate arginines in a monomethyl and di-methyl symmetric or asymmetric manner. Understanding the functional consequences and cataloging the methylated substrates of PRMTs have dramatically advanced the understanding of the PRMT enzymes and facilitated their linkage to disease2. Additionally, the development of potent and selective inhibitors has enabled chemical biology-based approaches to study PRMTs and has accelerated the clinical development of the PRMT targeting agents3. As the field of PRMTs progresses, novel methods and their applications will further enable elucidation of the regulation and biology of these enzymes.

In this methods collection, an article on the recombinant production of the PRMT proteins provides a detailed account of a baculovirus-based expression system4. Several PRMTs can be generated using the bacterial expression; however, larger or complex dependent PRMTs such as PRMT7 or PRMT5 require an insect cell expression system. This method has already benefited numerous in vitro efforts to determine the substrates of PRMTs.

Studying PRMT substrates in the cellular environment requires sophisticated detection approaches. Two articles in this collection present distinct methodologies that rely on antibody enrichment of methylated arginines followed by mass spectrometry quantitation or antibody-independent quantification using nuclear magnetic resonance (NMR). Several antibodies recognizing distinct methylation states were developed and have been used in studies identifying the PRMT substrates5. The mass spectrometry method enables the quantitative evaluation of cellular protein arginine methylation by employing metabolic labeling, subsequent sophisticated separation techniques, and antibody-based enrichment6. Several members of the PRMT family prefer methylating arginines that are found in repetitive RGG motifs residing in the intrinsically disordered regions of proteins7. The NMR spectroscopy-based method enables determining the methylation state and position of all the arginine derivatives (mono and di, symmetric and asymmetric) where they can be quantified reliably within complex biological samples8.

Finally, to identify methylated arginine residues in specific proteins, two articles provide approaches based on western blotting and the proximity ligation assay (PLA). Both of these methods utilize antibody-based detection but differ in the detection method and the information provided. The western blot method is quantitative and has been valuable in characterizing chemical probes9. The PLA provides information on the spatial location of the methylated arginine since it uses the target protein antibody and the generic antibody for mono or di-methyl arginine, thus circumventing the need to generate specific antibodies and can remarkably be used on fixed tissues10.

Together, these diverse methodologies will enable robust discovery of methylated arginines in various cell systems and disease states and further advance the fundamental knowledge of the biological function of the PRMT enzymes.

studying protein arginine methylation: approaches and methods

ARTICLES DISCUSSED:
Hutchinson, A., Seitova, A. Production of recombinant PRMT proteins using the Baculovirus expression vector system. Journal of Visualized Experiments. (173), doi: 10.3791/62510 (2021).
Maniaci, M., Marini, F., Massignani, E., Bonaldi, T. A mass spectrometry-based proteomics approach for global and high-confidence protein R-Methylation analysis. Journal of Visualized Experiments. (182), doi: 10.3791/62409 (2022).
Habisch, H., Zhang, F., Zhou, Q., Madl, T. Exploring the arginine methylome by nuclear magnetic resonance spectroscopy. Journal of Visualized Experiments. (178), doi: 10.3791/63245 (2021).
Szewczyk, M. M., Vu, V., Barsyte-Lovejoy, D. Quantitative methods to study protein arginine methyltransferase 1-9 activity in cells. Journal of Visualized Experiments. (174), doi: 10.3791/62418 (2021).
Poulard, C., Jacquemetton, J., Valantin, J., Treilleux, I., Le Romancer, M. Proximity ligation assay allows the detection, localization, and quantification of protein arginine methylation in fixed tissue. Journal of Visualized Experiments. (185), doi: 10.3791/64294 (2022).

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