Name: exploring the arginine methylome by nuclear magnetic resonance spectroscopy
Uploaded: 2021-12-16T14:00:00
Description: Watch this Scientific Journal Video about Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy at JoVE.com

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#8805;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 (D2O)</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 &#8805;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 (NaN3)</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 (Na2HPO4)</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>&#969;-NG-asymmetric dimethylarginine (ADMA)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-208093</td>
			<td></td>
		</tr>
		<tr>
			<td>&#969;-NG-monomethylarginine (MMA)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-200739A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#969;-NG-NG&#39;-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

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...

Watch this Scientific Journal Video about Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy (Video) | JoVE

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

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