Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Summary

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

Abstract

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.

Introduction

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 ω-(N^G,N^G)-asymmetric dimethylarginine (ADMA), ω-(N^G,N'^G)-symmetric dimethylarginine (SDMA), and ω-N^G-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 domains^8,9,10. In turn, this influences physiology and disease states, including cancer^11,12,13, 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 prognosis^21,22. 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 proteins^25,26. 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 technique^31,32. 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.

Protocol

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

1. 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 °C to minimize alcohol evaporation and increase sample stability.
2. 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.
3. 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).
4. 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.
5. 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.
6. Keep pipettes and the respective tips ready in the range of 10-1,000 µL.
   ​NOTE: Highly pure water, designated as H₂O, defined by a high resistance of ≥18.2 MΩ·cm^-1, was used throughout the work. It corresponds to double distilled water.

2. Sample collection and storage

1. Flash freeze the liquid samples (serum/plasma, cell culture supernatant, etc.) with liquid nitrogen and store them at -80 °C until usage.
2. Prepare the solid materials as mentioned below.
   1. 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.
   2. 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 °C. For further processing, see step 3.2.
   3. 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.
      ​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.

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.

1. For liquid samples, add 400 µL of ice-cold methanol to 200 µL 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.
2. In the case of solid materials, prepare enough 1.5 mL tubes for the lysates (use for centrifugation after homogenization).
   1. Carefully, but thoroughly, resuspend the cell pellets in 600 µL of MeOH/H₂O and transfer the entire liquid phase into the pulping tubes.
   2. Place tissues into pulping tubes (you may directly weigh them into the tubes) and add 600 µL of MeOH/H₂O (for 30-60 mg; adjust if weight differs substantially).
   3. 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).
   4. After homogenization, immediately put samples on the ice again and transfer the whole lysates into new 1.5 mL tubes.
3. Store samples for at least 30 min (up to several days) at -20 °C before further processing.
4. Centrifuge at 10,000 x g for 30 min at 4 °C. In the meantime, prepare enough 1.5 mL tubes to collect the supernatants.
   NOTE: This supernatant (designated "(1)" in Figure 1) might be discarded, be lyophilized, and directly processed for NMR (step 6) or analyzed otherwise.
5. 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 °C for a few days.

4. Protein hydrolysis

1. Add 500 µL 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.
2. 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 °C in a drying chamber.
3. Following hydrolysis, cool down the samples (~1 h).
4. After that, lyophilize overnight using a centrifuge with a high vacuum (below 100 Pa).
5. Dissolve the pellets - if completely dry (if not, continue lyophilization) - in 1 mL of 0.1 mol/L HCl and add 50 µL of chloroform (CHCl₃) to each tube; thoroughly dissolve the pellet with a 1,000 µL 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
6. Then, centrifuge at 8,000 x g for 10 min at room temperature.
7. 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).

5. Solid-phase extraction (SPE)

1. 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.
2. Manual SPE
   1. 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).
   2. 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.
   3. Then, apply a washing cycle as mentioned below.
      1. Add three times 1 mL of H₂O (each followed by centrifugation for 1 min at 800 x g at room temperature).
      2. 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).
      3. Add two times 1 mL of MeOH (each followed by centrifugation for 1 min at 800 x g at room temperature).
   4. 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).
3. 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.
   1. 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).
   2. 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.

6. Final preparation for NMR

1. Lyophilize samples overnight to complete dryness to get rid of ammonia and H₂O.
2. Dissolve each sample in 500 µL 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.
3. Record a 1D spectrum using the CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence^33,34 (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.
4. 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.
5. Perform absolute quantification by integrating the respective peak intensities of standards of a known concentration, e.g., 100 µmol/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.
6. 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 µL of d₆-DMSO.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL tubes	Greiner Bio One	188271
3-(trimethylsilyl) propionic acid-2,2,3,3-d4 sodium salt (TSP)	Alfa Aesar	A1448
5 mL tubes, round bottom	Greiner Bio One	115101
Ammonia Solution 32%	Roth	A990.1
Bruker 600 MHz NMR spectrometer, equipped with a TXI probe head	Bruker	-
Centrifuge, refrigerated, e.g. 5430 R	Eppendorf	5428000010
Chloroform ≥99% p.a.	Roth	3313.1
Cryocool	Thermo Scientific	SCC1	heat transfer fluid for SpeedVac System
Deuterium Oxide (D2O)	Cambridge Isotope Laboratories	DLM-10-PK
Dimethyl sulfoxide-d6 (d6-DMSO)	Cambridge Isotope Laboratories	DLM-6-1000
Drying Chamber	Binder	9090-0018
DURAN culture tubes, 13 x 100mm, GL 14, 9 mL	VWR International	212-0375
Edwards Deep vacuum oil pump RV5	Thermo Scientific	16234611	part of the SpeedVac System
Eppendorf 1.5 mL tubes	Greiner Bio One	616201
Gilson pipetting robot GX-241 Aspec	Gilson Inc.	26150008
L-arginine	AppliChem	A3675
Methanol ≥99%	Roth	8388.4
Milli-Q water aparatus	Millipore	ZIQ7000T0
Oasis MCX 1cc/30 mg, 1 mL cartridges	Waters	186000252	https://www.waters.com/waters/en_US/Waters-Oasis-Sample-Extraction-SPE-Products/
Phosphate Buffered Saline (PBS)	Lonza	LONBE17-512F
Precellys 24 tissue homogenizer	Bertin Instruments	P000669-PR240-A	https://www.bertin-instruments.com/product/sample-preparation-homogenizers/precellys24-tissue-homogenizer/
Precellys tubes (pulping tubes)	VWR International	432-0351
Precellyse 1.4 mm zirconium oxide beads	VWR International	432-0356
Reacti-Therm/ReactiVap Heating, Stirring, and Evaporation Modules	Thermo Scientific	TS-18820	https://www.thermofisher.com/order/catalog/product/TS-18820
Rotor for 1.5 mL tubes, FA-45-30-11	Eppendorf	5427753001
Savant Refrigerated Cooling Trap	Thermo Scientific	15996161	part of the SpeedVac System
Savant SpeedVac vacuum concentrator SPD210	Thermo Scientific	15906181	part of the SpeedVac System; equipped with rotor for 1.5 ml tubes
Screw caps for glas vials with PTFE sealing, DN9	Dr. R. Forche Chromatographie	CT11B3011
Seasand	Roth	8441.3
Short thread glas vials 1.5 mL, ND9	Dr. R. Forche Chromatographie	VT1100309
Sodium azide (NaN3)	Roth	K305.1
Sodium hydroxide (NaOH)	VWR	BDH7363-4
Sodium phosphate dibasic (Na2HPO4)	VWR	80731-078
TopSpin 4.0 (Software)	Bruker	-	https://www.bruker.com
ω-NG-asymmetric dimethylarginine (ADMA)	Santa Cruz Biotechnology	sc-208093
ω-NG-monomethylarginine (MMA)	Santa Cruz Biotechnology	sc-200739A
ω-NG-NG'-symmetric dimethylarginine (SDMA)	Santa Cruz Biotechnology	sc-202235A