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 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...

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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 ...

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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

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

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.

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the mechanical properties of the cells is often challenging. Conventional measurement methods based on contact such as compression or stretching are prone to cause cell damage, affecting measurement accuracy and subsequent cell culture. Measurements in adherent state can also affect accuracy, especially after irradiation since ionizing radiation will flatten cells and enhance adhesion. Here, a cell mechanics measurement system based on acoustofluidic method has been developed. The cell compressibility can be obtained by recording the cell motion trajectory under the action of the acoustic force, which can realize fast and non-destructive measurement in suspended state. This paper reports in detail the protocols for chip design, sample preparation, trajectory recording, parameter extraction and analysis. The compressibility of different types of tumor cells was measured based on this method. Measurement of the compressibility of nucleus was also achieved by adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel. Combined with the molecular level verification of immunofluorescence experiments, the cell compressibility before and after drug-induced epithelial to mesenchymal transition (EMT) were compared. Further, the change of cell compressibility after X-ray irradiation with different doses was revealed. The cell mechanics measurement method proposed in this paper is universal and flexible and has broad application prospects in scientific research and clinical practice.

Here a protocol is presented to build a fast and non-destructive system for measuring cell or nucleus compressibility based on acoustofluidic microdevice. Changes in mechanical properties of tumor cells after epithelial-mesenchymal transition or ionizing radiation were investigated, demonstrating the application prospect of this method in scientific research and clinical practice.

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

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Hyperpolarized ¹³C Metabolic Magnetic Resonance Spectroscopy and Imaging

In Vivo EPR Assessment of pH, pO₂, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment