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)

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....

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

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