Name: Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy (Video) | JoVE Journal
Uploaded: 2021-12-16T14:00:00.000+00:00
Duration: 7 min 2 s
Description: Learn to quantify arginine methylation dynamics using 1H-NMR spectroscopy across cells, tissues, and biofluids. This method enables biomarker discovery for cancer research and patient stratification.

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5 minutes

The work was supported by Austrian Science Fund (FWF) grants P28854, I3792, doc.funds BioMolStruct DOC 130, DK-MCD W1226 BioTechMed-Graz (Flagship project DYNIMO), Austrian Research Promotion Agency (FFG) grants 864690 and 870454, the Integrative Metabolism Research Center Graz; Austrian Infrastructure Program 2016/2017, the Styrian Government (Zukunftsfonds) and Startup Fund for High-level Talents of Fujian Medical University (XRCZX2021020). We thank the Center of Medical Research for access to cell culture facilities. F.Z. was trained within the frame of the PhD program Molecular Medicine, Medical University of Graz. Q.Z. was trained within the frame of the PhD program Metabolic and Cardiovascular Diseases, Medical University of Graz.

Yeast protein hydrolysates were used as samples for the Representative results of this work. The entire protocol is summarized in Figure 1.
1. Preparation of materials and reagents
<ol>
	<li>Methanol/water: prepare a mixture of two parts of 99% methanol (MeOH) and one part of H2O, designated as MeOH/H2O. Store pure MeOH and MeOH/H2O at -20 &#176;C to minimize alcohol evaporation and increase sample stability.</li>
	<li>Hydrochloric acid (HCl): dilute 9 mol/L from concentrated HCl (12.0 mol/L or 37%) as well as 0.1 mol/L of HCl. The higher concentrated (9 mol/L) solution is used for protein hydrolysis, while the lower concentrated (0.1 mol/L) one is used for solid-phase extraction. 
	CAUTION: Work inside the fume hood.</li>
	<li>Prepare a Replacement solution for SPE containing 10% of a saturated ammonia solution (stock: 30% w/w ammonia), 50% of methanol, and 40% of water (v/v).</li>
	<li>NMR Buffer: Dissolve 5.56 g of disodium hydrogen phosphate (Na2HPO4, 0.08 mol/L), 0.4 g of 3-(trimethylsilyl) propionic acid-2,2,3,3-d4 sodium salt (TSP, 5 mmol/L), 0.04 % (w/v) of sodium azide (NaN3) in 500 mL of deuterium dioxide (D2O) and adjust to pH 7.4 (using HCl or NaOH, respectively) (see Table of Materials). Check the purity of each new buffer batch by NMR spectroscopy. 
	NOTE: The amount of any contaminants (e.g., ethanol) should be as low as possible.</li>
	<li>Fill pulping tubes with zirconium oxide beads (2 mL tubes and 1.4 mm beads are suitable for most applications, but different variants are available) (see Table of Materials). Fill roughly 10-20 beads by hand or buy pre-filled tubes, which are also available.</li>
	<li>Keep pipettes and the respective tips ready in the range of 10-1,000 &#181;L. 
	&#8203;NOTE: Highly pure water, designated as H2O, defined by a high resistance of &#8805;18.2 M&#937;&#183;cm-1, was used throughout the work. It corresponds to double distilled water.</li>
</ol>
2. Sample collection and storage
<ol>
	<li>Flash freeze the liquid samples (serum/plasma, cell culture supernatant, etc.) with liquid nitrogen and store them at -80 &#176;C until usage.</li>
	<li>Prepare the solid materials as mentioned below.
	<ol>
		<li>Collect the cells from cell cultures (3-5 million cells) either by collecting adherent cells, after washing with cold phosphate-buffered saline, scraping and centrifugation, or direct centrifugation of cells grown in suspension.</li>
		<li>Remove the cell supernatants (which might be collected for further analysis, either by consecutive direct measurement, see step 6, or after precipitation of soluble proteins, see step 3.1), and then flash freeze the pellets with liquid nitrogen and store at -80 &#176;C. For further processing, see step 3.2.</li>
		<li>Store and collect the tissues as per the aim of the study. For very homogenous tissues, such as the liver or muscle, analyze any part of it. For example, in the case of a mouse brain, determine global arginine methylation of the entire brain or brain sections. 
		&#8203;NOTE: The ideal weight of tissues to be processed is 30-60 mg. It is advisable to freeze and pulverize the entire brain and use aliquots thereof. Alternatively, specific brain parts (e.g., frontal, cerebellum, occipital regions, or one hemisphere, weighing ~250 mg) may be used.</li>
	</ol>
	</li>
</ol>
3. Preliminary sample preparation
NOTE: Perform the work on ice, keeping MeOH cold. The samples must also be kept on ice to avoid protein degradation.
<ol>
	<li>For liquid samples, add 400 &#181;L of ice-cold methanol to 200 &#181;L of the sample. Continue with step 3.3. 
	NOTE: If less sample is available, adjust the volumes accordingly; NMR sensitivity is usually high enough for smaller sample amounts, but has to be tested individually.</li>
	<li>In the case of solid materials, prepare enough 1.5 mL tubes for the lysates (use for centrifugation after homogenization).
	<ol>
		<li>Carefully, but thoroughly, resuspend the cell pellets in 600 &#181;L of MeOH/H2O and transfer the entire liquid phase into the pulping tubes.</li>
		<li>Place tissues into pulping tubes (you may directly weigh them into the tubes) and add 600 &#181;L of MeOH/H2O (for 30-60 mg; adjust if weight differs substantially).</li>
		<li>Put the tubes into the tissue homogenizer (see Table of Materials) and either homogenize one time for 20 s with heavy shaking (for cell pellets, soft tissues) or two times for 20 s each (or longer if necessary) with an interval of 5 min in between (for stiff tissues).</li>
		<li>After homogenization, immediately put samples on the ice again and transfer the whole lysates into new 1.5 mL tubes.</li>
	</ol>
	</li>
	<li>Store samples for at least 30 min (up to several days) at -20 &#176;C before further processing.</li>
	<li>Centrifuge at 10,000 x g for 30 min at 4 &#176;C. In the meantime, prepare enough 1.5 mL tubes to collect the supernatants. 
	NOTE: This supernatant (designated &#34;(1)&#34; in Figure 1) might be discarded, be lyophilized, and directly processed for NMR (step 6) or analyzed otherwise.</li>
	<li>In this protocol, the MeOH precipitate (= pellet obtained after step 3.1 or 3.2.2, respectively) is used for further analysis containing, among other things, components such as nucleic acids and lipids, arginine-methylated proteins. Continue with protein hydrolysis or store the pellets at -20 &#176;C for a few days.</li>
</ol>
4. Protein hydrolysis
<ol>
	<li>Add 500 &#181;L of 9 mol/L of HCl to each sample. Then, truncate the caps of each tube with scissors and place them into the glass culture tubes (Figure 2A). Carefully, but tightly, close the red caps (thereby checking the sealing). 
	NOTE: Before use, always check each sealing (gray PTFE foil inside the red screw caps) for proper tightening and regularly clean them with water.</li>
	<li>When finished, place the tubes into a beaker partly filled with sand (for better heat transfer) and hydrolyze samples for about 16 h at 110 &#176;C in a drying chamber.</li>
	<li>Following hydrolysis, cool down the samples (~1 h).</li>
	<li>After that, lyophilize overnight using a centrifuge with a high vacuum (below 100 Pa).</li>
	<li>Dissolve the pellets - if completely dry (if not, continue lyophilization) - in 1 mL of 0.1 mol/L HCl and add 50 &#181;L of chloroform (CHCl3) to each tube; thoroughly dissolve the pellet with a 1,000 &#181;L pipette, thereby mixing the liquids, and then transfer the complete volume into a new tube. 
	CAUTION: Always work with small amounts to minimize extensive evaporation</li>
	<li>Then, centrifuge at 8,000 x g for 10 min at room temperature.</li>
	<li>Carefully collect the upper phase of the biphasic liquid containing the water-soluble fraction (Figure 2B, the fraction with the lower density) with a pipette and fill it into new tubes. Use 1.5 mL tubes for manual SPE (section 5.2) or glass vials for robotic SPE (section 5.3 and Figure 2D). Avoid spilling over CHCl3 and its contents (Figure 2C).</li>
</ol>
5. Solid-phase extraction (SPE)
<ol>
	<li>Before the first usage, clean the SPE cartridges two times with 1 mL of the replacement solution (step 1.3) followed by centrifugation at 800 x g for 1 min at room temperature (placed into 15 mL tubes). They can be used 10-20 times each.</li>
	<li>Manual SPE
	<ol>
		<li>Pre-condition the cartridges (see Table of Materials) in every run with 1 mL of pure methanol and 2 x 1 mL of PBS, each time followed by centrifugation at 800 x g for 1 min at room temperature (placed into 15 mL tubes).</li>
		<li>After that, load the samples (from step 4.7) onto the SPE cartridges and centrifuge them at 600 x g for 2 min at room temperature.</li>
		<li>Then, apply a washing cycle as mentioned below.
		<ol>
			<li>Add three times 1 mL of H2O (each followed by centrifugation for 1 min at 800 x g at room temperature).</li>
			<li>Add five times 1 mL of 0.1 mol/L HCl (each followed by centrifugation for 1 min at 800 x g at room temperature).</li>
			<li>Add two times 1 mL of MeOH (each followed by centrifugation for 1 min at 800 x g at room temperature).</li>
		</ol>
		</li>
		<li>Then, eluate arginine and its derivatives with 3 x 1 mL of replacement solution (followed by centrifugation 1 min at 800 x g at room temperature) into a single 15 mL tube. 
		NOTE: The replacement solution serves as eluent for arginine and derivatives and is also used for cleaning the cartridges (all substances are either washed out before elution or at the basic pH value of the replacement solution). Still, to avoid contamination over time, the re-usage should be limited (see step 5.1).</li>
	</ol>
	</li>
	<li>Robotic SPE 
	NOTE: Here, a pipetting robot (the main part consisting of a pipetting needle, a washing station for the needle, a reagent supply, and positions for samples and eluates, see Table of Materials) is supplied with a cartridge holder for the SPE columns. For other instruments, make sure to be fully equipped with all things listed.
	<ol>
		<li>Fill the supernatant (from step 4.7) directly into glass vials with PTFE sealing (Figure 2D), prepare enough reagents (100 mL of each, i.e., replacement solution, 99% of MeOH, PBS, H2O, and 0.1 of mol/L HCl), 5 mL tubes for elution and washing solution for the needle of the robot (usually H2O).</li>
		<li>Use an application or method in the software of the robot based precisely on the steps described for manual SPE (step 5.2). 
		NOTE: The details may vary a lot among instrument suppliers, so refer to the software manual of the respective robot for programming. A pipetting robot usually works by applying air pressure onto the cartridges instead of centrifugation, which is the main difference. If the protocol is established and run for the first time, include controls (e.g., L-arginine of known concentration) to verify an optimal workflow.</li>
	</ol>
	</li>
</ol>
6. Final preparation for NMR
<ol>
	<li>Lyophilize samples overnight to complete dryness to get rid of ammonia and H2O.</li>
	<li>Dissolve each sample in 500 &#181;L of NMR buffer (step 1.4) and transfer to NMR tubes. Importantly, the volume must be the same in all tubes, and samples must be homogenous (free of residues and lipids, which tend to aggregate). 
	NOTE: The details of NMR spectroscopy go beyond this method review and are described in more detail elsewhere27. Here, only the main requirements and steps are explained. The quantification of arginine and its metabolites are performed on a 600 MHz NMR spectrometer (see Table of Materials). In principle, any other NMR spectrometer/NMR field strengths can be used, provided that the NMR signals of arginine and methylated arginines can be detected with sufficient sensitivity and without signal overlap. Any probe head with z-axis gradients able to record 1H-spectra can be used, provided that the pulse sequences are set up accordingly.</li>
	<li>Record a 1D spectrum using the CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence33,34 (cpmgpr1d, 512 scans, size of fid 73728, 11904.76 Hz spectral width on a 600 MHz NMR, recycle delay 4 s) with water signal suppression using presaturation.</li>
	<li>In these experiments, remove the 1H scalar couplings by virtual decoupling, reducing signal overlap. Record additional for specific questions and/or complex samples or matrices, e.g., 1H-13C-heteronuclear single quantum coherence (HSQC) spectra27. 
	NOTE: For more complex matrices in which 1H NMR signals of methylated arginines could be partially overlapped with 1H NMR signals of other metabolites (e.g., lysine), 1H homo-nuclear J-resolved spectra (JRES)35 are required.</li>
	<li>Perform absolute quantification by integrating the respective peak intensities of standards of a known concentration, e.g., 100 &#181;mol/arginine. 
	NOTE: Routinely, ADMA, SDMA, and MMA (see Table of Materials) are reported as a ratio relative to arginine. This has a significant advantage that no separate normalization (e.g., cell number, tissue mass, protein concentrations) needs to be performed. In that case, arginine serves as an internal standard, and relative quantification is sufficient to gain biologically relevant information.</li>
	<li>For differentiation of SDMA and MMA, lyophilize the samples again be to get rid of D2O (if they have been resuspended in NMR buffer before), which is then replaced by deuterated dimethyl sulfoxide (d6-DMSO), enabling the resolution of methyl resonances27. Therefore, aspirate the samples from the NMR tubes with Pasteur pipettes, lyophilize, and dissolve them in 500 &#181;L of d6-DMSO.</li>
</ol>

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<li>Davids, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+determination+of+asymmetric+and+symmetric+dimethylarginine%2C+L-monomethylarginine%2C+L-arginine%2C+and+L-homoarginine+in+biological+samples+using+stable+isotope+dilution+liquid+chromatography+tandem+mass+spectrometry%5BTitle%5D)+AND+%22Journal+of+Chromatography+B%3A+Analytical+Technologies+in+the+Biomedical+and+Life+Sciences%22%5BJournal%5D)">Simultaneous determination of asymmetric and symmetric dimethylarginine, L-monomethylarginine, L-arginine, and L-homoarginine in biological samples using stable isotope dilution liquid chromatography tandem mass spectrometry.</a> Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 900, 38-47 (2012).</li>
<li>Stryeck, S., Birner-Gruenberger, R., Madl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+metabolomics+as+emerging+tool+to+study+autophagy+regulation%5BTitle%5D)+AND+%22Microbial+Cell%22%5BJournal%5D)">Integrative metabolomics as emerging tool to study autophagy regulation.</a> Microbial Cell. 4 (8), 240-258 (2017).</li>
<li>Vignoli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+metabolomics+by+1D+NMR%5BTitle%5D)+AND+%22Angewandte+Chemie+International+Edition%22%5BJournal%5D)">High-throughput metabolomics by 1D NMR.</a> Angewandte Chemie International Edition. 58 (4), 968-994 (2019).</li>
<li>Carr, H. Y., Purcell, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+diffusion+on+free+precession+in+nuclear+magnetic+resonance+experiments%5BTitle%5D)+AND+%22Physical+Review%22%5BJournal%5D)">Effects of diffusion on free precession in nuclear magnetic resonance experiments.</a> Physical Review. 94 (3), 630-638 (1954).</li>
<li>Meiboom, S., Gill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modified+spin-echo+method+for+measuring+nuclear+relaxation+times%5BTitle%5D)+AND+%22Review+of+Scientific+Instruments%22%5BJournal%5D)">Modified spin-echo method for measuring nuclear relaxation times.</a> Review of Scientific Instruments. 29 (8), 688-691 (1958).</li>
<li>Nagayama, K., Wuthrich, K., Bachmann, P., Ernst, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-dimensional+J-resolved+1H+n.m.r.+spectroscopy+for+studies+of+biological+macromolecules%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Two-dimensional J-resolved 1H n.m.r. spectroscopy for studies of biological macromolecules.</a> Biochemical and Biophysical Research Communications. 78 (1), 99-105 (1977).</li>
<li>Stryeck, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum+concentrations+of+Citrate%2C+Tyrosine%2C+2-+and+3-+Hydroxybutyrate+are+associated+with+increased+3-month+mortality+in+acute+heart+failure+patients%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Serum concentrations of Citrate, Tyrosine, 2- and 3- Hydroxybutyrate are associated with increased 3-month mortality in acute heart failure patients.</a> Scientific Reports. 9 (1), 6743(2019).</li>
<li>Zhang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-specific+landscape+of+metabolic+dysregulation+during+ageing%5BTitle%5D)+AND+%22Biomolecules%22%5BJournal%5D)">Tissue-specific landscape of metabolic dysregulation during ageing.</a> Biomolecules. 11 (2), (2021).</li>
<li>Zhang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growing+human+hepatocellular+tumors+undergo+a+global+metabolic+reprogramming%5BTitle%5D)+AND+%22Cancers%22%5BJournal%5D)">Growing human hepatocellular tumors undergo a global metabolic reprogramming.</a> Cancers. 13 (8), (2021).</li>
<li>Pahlich, S., Zakaryan, R. P., Gehring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+arginine+methylation%3A+Cellular+functions+and+methods+of+analysis%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Protein arginine methylation: Cellular functions and methods of analysis.</a> Biochimica et Biophysica Acta. 1764 (12), 1890-1903 (2006).</li>
<li>Habisch, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroectodermally+converted+human+mesenchymal+stromal+cells+provide+cytoprotective+effects+on+neural+stem+cells+and+inhibit+their+glial+differentiation%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Neuroectodermally converted human mesenchymal stromal cells provide cytoprotective effects on neural stem cells and inhibit their glial differentiation.</a> Cytotherapy. 12 (4), 491-504 (2010).</li>
<li>Ibanez, G., McBean, J. L., Astudillo, Y. M., Luo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+enzyme-coupled+ultrasensitive+luminescence+assay+for+protein+methyltransferases%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases.</a> Analytical Biochemistry. 401 (2), 203-210 (2010).</li>
<li>Hevel, J. M., Price, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+direct+measurement+of+methyltransferase+activity+in+about+30min%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Rapid and direct measurement of methyltransferase activity in about 30min.</a> Methods. 175, 3-9 (2020).</li>
<li>Altincekic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Site-specific+detection+of+arginine+methylation+in+highly+repetitive+protein+motifs+of+low+sequence+complexity+by+NMR%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Site-specific detection of arginine methylation in highly repetitive protein motifs of low sequence complexity by NMR.</a> Journal of the American Chemical Society. 142 (16), 7647-7654 (2020).</li>
<li>Kaneb, H. M., Dion, P. A., Rouleau, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+FUS+about+arginine+methylation+in+ALS+and+FTLD%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">The FUS about arginine methylation in ALS and FTLD.</a> The EMBO Journal. 31 (22), 4249-4251 (2012).</li>
</ol>

The authors declare no competing interests.

In the following section, the primary focus lies on the method itself; the biological implications of arginine methylation are described in the Introduction section.
Firstly, tissues of different stiffness might need adjustment of sample lysis: cells from cell culture (including bacteria, yeast, etc.) and tissues like brain, young liver, smooth muscle, etc., can quickly be homogenized. For tissues of high stiffness (including liver of elderly subjects, arteries, bones, etc.), the homogenizatio...

During the last two decades, methylation of arginine residues has been recognized as an essential posttranslational modification of proteins. It affects fundamental biological processes like regulation of transcription, signal transduction, and many more1. The main proteins involved in the regulation of arginine methylation are protein arginine methyltransferases (PRMTs)2. The main derivatives of arginine are &#969;-(NG,NG)-asymmetric dimethylarginine (ADMA), &#969;-(NG,N&#39;G)-symmetric dimethylarginine (SDMA), and &#969;-NG-monomethylarginine (MMA)2.
PRMTs use S-adenosyl-l-methionine to transfer methyl groups to the terminal guanidino group (with two equivalent amino groups) of protein-bound arginine1. Two main enzymes can be distinguished: Both type I and type II enzymes catalyze the first methylation step to form MMA (which thereby loses its symmetry). Following this step, type I enzymes (e.g., PRMT1, 2, 3, 4, 6, 8) use MMA as the substrate to form ADMA, whereas type II enzymes (primarily PRMT5 and PRMT9) produce SDMA. PRMT1 was the first protein arginine methyltransferase to be isolated from mammalian cells3. Still, PRMTs have been evolutionarily conserved4 in other animals like non-mammalian vertebrates, invertebrate chordates, echinoderms, arthropods, and nematodes cnidarians5, plants6, and protozoa, including fungi like yeast7. In many cases, knockout of one of the PRMTs leads to loss of viability, revealing the essential role of methylated arginine species involved in fundamental cellular processes like transcription, translation, signal transduction, apoptosis, and liquid-liquid phase separation (meaning the formation of membrane-less organelles, e.g., nucleoli), which regularly involves arginine-rich domains8,9,10. In turn, this influences physiology and disease states, including cancer11,12,13, multiple myeloma14, cardiovascular diseases15, viral pathogenesis, spinal muscular atrophy16, diabetes mellitus17, and aging1. Increased ADMA levels in the bloodstream, e.g., derived from lung18 due to protein breakdown, are thought to be connected with endothelial dysfunction, chronic pulmonary disease19, and other syndromes of cardiovascular disease20. Overexpression of PRMTs has been found to accelerate tumorigenesis and is associated with poor prognosis21,22. Besides, ablation of PRMT6 and PRMT7 triggers a cellular senescence phenotype23. Significant decreased ADMA and PRMT1 have been found during the aging of WI-38 fibroblasts24.
The challenge is understanding how methylation acts in (patho)physiological processes is identifying and quantifying protein arginine methylation. Most of the current approaches use antibodies to detect methylated arginines. However, these antibodies are still context-specific and might fail to recognize different motifs of arginine methylated proteins25,26. In the described protocol, all of the arginine derivatives mentioned afore can be quantified reliably by nuclear magnetic resonance (NMR) spectroscopy, i.e., alone, in combination, or, as in most cases, within complex biological matrices like eukaryotic cells (e.g., from yeast, mouse, or human origin) and tissues27, as well as serum28. For proteins and those complex matrices, protein hydrolysis29 is a prerequisite to generate free (modified) amino acids, such as arginine, MMA, SDMA, and ADMA. Solid-phase extraction (SPE)30 enables the enrichment of the compounds of interest. Finally, 1H-NMR spectroscopy allows the parallel detection of arginine and all the major methyl derivatives of arginine. NMR spectroscopy comes with the advantage that it is genuinely quantitative, highly reproducible, and a robust technique31,32. The final NMR measurements can be done afterward when many samples have been collected and prepared. Finally, this protocol mainly focuses on sample preparation as this does not require an own NMR spectrometer. It can be performed in most biochemical laboratories. Still, some hints on which NMR spectroscopy measurements should be done are provided in this work.

<table><tbody><tr><td>15 mL tubes</td><td>Greiner Bio One</td><td>188271</td><td></td></tr><tr><td>3-(trimethylsilyl) propionic acid-2,2,3,3-d4 sodium salt (TSP)</td><td>Alfa Aesar</td><td>A1448</td><td></td></tr><tr><td>5 mL tubes, round bottom</td><td>Greiner Bio One</td><td>115101</td><td></td></tr><tr><td>Ammonia Solution 32%</td><td>Roth</td><td>A990.1</td><td></td></tr><tr><td>Bruker 600 MHz NMR spectrometer, equipped with a TXI probe head</td><td>Bruker</td><td>-</td><td></td></tr><tr><td>Centrifuge, refrigerated, e.g. 5430 R</td><td>Eppendorf</td><td>5428000010</td><td></td></tr><tr><td>Chloroform &amp;ge;99% p.a.</td><td>Roth</td><td>3313.1</td><td></td></tr><tr><td>Cryocool</td><td>Thermo Scientific</td><td>SCC1</td><td>heat transfer fluid for SpeedVac System</td></tr><tr><td>Deuterium Oxide (D&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Cambridge Isotope Laboratories</td><td>DLM-10-PK</td><td></td></tr><tr><td>Dimethyl sulfoxide-d6 (d6-DMSO)</td><td>Cambridge Isotope Laboratories</td><td>DLM-6-1000</td><td></td></tr><tr><td>Drying Chamber</td><td>Binder</td><td>9090-0018</td><td></td></tr><tr><td>DURAN culture tubes, 13 x 100mm, GL 14, 9 mL</td><td>VWR International</td><td>212-0375</td><td></td></tr><tr><td>Edwards Deep vacuum oil pump RV5</td><td>Thermo Scientific</td><td>16234611</td><td>part of the SpeedVac System</td></tr><tr><td>Eppendorf 1.5 mL tubes</td><td>Greiner Bio One</td><td>616201</td><td></td></tr><tr><td>Gilson pipetting robot GX-241 Aspec</td><td>Gilson Inc.</td><td>26150008</td><td></td></tr><tr><td>L-arginine</td><td>AppliChem</td><td>A3675</td><td></td></tr><tr><td>Methanol &amp;ge;99%</td><td>Roth</td><td>8388.4</td><td></td></tr><tr><td>Milli-Q water aparatus</td><td>Millipore</td><td>ZIQ7000T0</td><td></td></tr><tr><td>Oasis MCX 1cc/30 mg, 1 mL cartridges</td><td>Waters</td><td>186000252</td><td>https://www.waters.com/waters/en_US/Waters-Oasis-Sample-Extraction-SPE-Products/</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Lonza</td><td>LONBE17-512F</td><td></td></tr><tr><td>Precellys 24 tissue homogenizer</td><td>Bertin Instruments</td><td>P000669-PR240-A</td><td>https://www.bertin-instruments.com/product/sample-preparation-homogenizers/precellys24-tissue-homogenizer/</td></tr><tr><td>Precellys tubes (pulping tubes)</td><td>VWR International</td><td>432-0351</td><td></td></tr><tr><td>Precellyse 1.4 mm zirconium oxide beads</td><td>VWR International</td><td>432-0356</td><td></td></tr><tr><td>Reacti-Therm/ReactiVap Heating, Stirring, and Evaporation Modules</td><td>Thermo Scientific</td><td>TS-18820</td><td>https://www.thermofisher.com/order/catalog/product/TS-18820</td></tr><tr><td>Rotor for 1.5 mL tubes, FA-45-30-11</td><td>Eppendorf</td><td>5427753001</td><td></td></tr><tr><td>Savant Refrigerated Cooling Trap</td><td>Thermo Scientific</td><td>15996161</td><td>part of the SpeedVac System</td></tr><tr><td>Savant SpeedVac vacuum concentrator SPD210</td><td>Thermo Scientific</td><td>15906181</td><td>part of the SpeedVac System; equipped with rotor for 1.5 ml tubes</td></tr><tr><td>Screw caps for glas vials with PTFE sealing, DN9</td><td>Dr. R. Forche Chromatographie</td><td>CT11B3011</td><td></td></tr><tr><td>Seasand</td><td>Roth</td><td>8441.3</td><td></td></tr><tr><td>Short thread glas vials 1.5 mL, ND9</td><td>Dr. R. Forche Chromatographie</td><td>VT1100309</td><td></td></tr><tr><td>Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td>Roth</td><td>K305.1</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>VWR</td><td>BDH7363-4</td><td></td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>VWR</td><td>80731-078</td><td></td></tr><tr><td>TopSpin 4.0 (Software)</td><td>Bruker</td><td>-</td><td>https://www.bruker.com</td></tr><tr><td>&amp;omega;-NG-asymmetric dimethylarginine (ADMA)</td><td>Santa Cruz Biotechnology</td><td>sc-208093</td><td></td></tr><tr><td>&amp;omega;-NG-monomethylarginine (MMA)</td><td>Santa Cruz Biotechnology</td><td>sc-200739A</td><td></td></tr><tr><td>&amp;omega;-NG-NG&#039;-symmetric dimethylarginine (SDMA)</td><td>Santa Cruz Biotechnology</td><td>sc-202235A</td><td></td></tr></tbody></table>

exploring the arginine methylome by nuclear magnetic resonance spectroscopy

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

Routinely, 1H 1D projections of 2D J-resolved (JRES), virtually decoupled NMR spectra are used for peak assignments and quantifications in our laboratory36. Figure 3 shows representative JRES spectra of yeast protein hydrolysates purified using the present SPE protocol. Though at very different concentrations, both substances can be separated and quantified in a cellular matrix. Based on the number of protons of the specific methyl (-CH3) or meth...

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

exploring-arginine-methylome-nuclear-magnetic-resonance

Research

JoVE Journal

Cancer Research

Two methods for assigning the α- and ε-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro Methylation Assay to Study Protein Arginine Methylation

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

Here, we present a protocol detailing the acquisition, processing, and analysis of a series of NMR experiments aimed at characterizing protein-glycan interactions in solution. Most common ligand-based and protein-based methodologies are outlined, which undoubtedly contribute to the fields of structural glycobiology and molecular recognition studies.

Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue

Here, we present a protocol to semi-quantitatively assess global m⁶A levels using dot blot. Total RNA is extracted, denatured, spotted on a nylon membrane, probed with anti-m⁶A antibody, and visualized by chemiluminescence. Signal intensity, quantified by ImageJ grayscale analysis, reflects relative methylation abundance, providing a reproducible workflow for research.

Dot Blot Assay for Detecting Global N6-Methyladenosine RNA Modification Levels

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Learn to quantify arginine methylation dynamics using 1H-NMR spectroscopy across cells, tissues, and biofluids. This method enables biomarker discovery for cancer research and patient stratification.

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

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