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>

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

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

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

1.3K Views.  Medical University of Graz. The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Video: Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

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

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

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

Two methods for assigning the &#945;- and &#949;-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.

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

Chemistry

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

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.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Biochemistry

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.

Proximity ligation assay is a very useful technique to localize and quantify arginine methylation of a given protein when the modified arginine residue is unknown and/or if no specific antibody is available.

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often ...

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

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

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.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not only provides quantitative data but also permits structural identification of the metabolites present in complex mixtures.
Metabolic profiling by 1H NMR has proven useful in the study of various types of plant scenarios, which include the evaluation of crop&#160;conditions, harvest and post- harvest treatments, metabolic phenotyping, metabolic pathways, gene regulation, identification of biomarkers, chemotaxonomy, quality control, denomination of origin, among others. However, signal overlapping of the large number of resonances with expanded J-coupling multiplicities complicates the spectra analysis and its interpretation, and represents a limitation for classical 1H NMR profiling.
In the last decade, novel NMR broadband homonuclear decoupling techniques through which multiplet signals collapse into single resonance lines - commonly called Pure Shift methods - have been developed to overcome the spectra resolution problem inherent to 1H NMR classical spectra.
Here a step-by-step protocol of the plant extract preparation and the procedure to record optimal Pure Shift PSYCHE and SAPPHIRE-PSYCHE spectra in three different plant matrices - Vanilla plant&#160;leaves, potato tubers (S. tuberosum), and Cape gooseberries (P. peruviana) - is presented. The effect of the gain in resolution in metabolic identification, correlation analysis and multivariate analyses, as compared against classical spectra, is discussed.

This paper presents the use of PSYCHE and SAPPHIRE-PSYCHE in the metabolic profiling of plants and includes detailed procedures for sample preparation and optimal Pure Shift NMR spectra recording. Examples through which the gain in resolution achieved by homonuclear decoupling allows a more comprehensive understanding of the system are discussed.

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Neuroscience

Advanced NMR Techniques for Glycan-Protein Interaction Analysis

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

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and their biological consequences are intimately related to the three-dimensional structures of both partners, as well as to their dynamic features and their presentation on the corresponding cell compartments. NMR techniques are unique to disentangle these characteristics and, indeed, diverse NMR-based methodologies have been developed and applied to monitor the binding events of glycans with their associate receptors. This protocol outlines the procedures to acquire, process and analyze two of the most powerful NMR methodologies employed in the NMR-glycobiology field, 1H-Saturation transfer difference (STD) and 1H,15N-Heteronuclear single quantum coherence (HSQC) titration experiments, which complementarily offer information from the glycan and protein perspective, respectively. Indeed, when combined they offer a powerful toolkit for elucidating both the structural and dynamic aspects of molecular recognition processes. This comprehensive approach enhances our understanding of glycan-protein interactions and contributes to advancing research in the chemical glycobiology field.

Author Spotlight: Unveiling the Structural and Dynamic Aspects of Glycan Molecular Recognition

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.

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

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.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Biology

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

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

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

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.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

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

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

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.

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

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Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

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

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

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

In vitro Methylation Assay to Study Protein Arginine Methylation

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

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