Electrophoretic Determination of Symmetric and Asymmetric Dimethylarginine in Human Blood Plasma with Whole Capillary Sample Injection.

Petr Tůma,Dušan Koval,François Couderc,Blanka Sommerová

doi:10.3390/ijms22062970

Abstract

Asymmetric and symmetric dimethylarginines are toxic non-coded amino acids. They are formed by post-translational modifications and play multifunctional roles in some human diseases. Their determination in human blood plasma is performed using capillary electrophoresis with contactless conductivity detection. The separations are performed in a capillary covered with covalently bonded PAMAPTAC polymer, which generates anionic electroosmotic flow and the separation takes place in the counter-current regime. The background electrolyte is a 750 mM aqueous solution of acetic acid with pH 2.45. The plasma samples for analysis are treated by the addition of acetonitrile and injected into the capillary in a large volume, reaching 94.5% of the total volume of the capillary, and subsequently subjected to electrophoretic stacking. The attained LODs are 16 nm for ADMA and 22 nM for SDMA. The electrophoretic resolution of both isomers has a value of 5.3. The developed method is sufficiently sensitive for the determination of plasmatic levels of ADMA and SDMA. The determination does not require derivatization and the individual steps in the electrophoretic stacking are fully automated. The determined plasmatic levels for healthy individuals vary in the range 0.36–0.62 µM for ADMA and 0.32–0.70 µM for SDMA.

Highlights

N-methylation of the amino acid L-arginine in proteins is a common post-translation modification, which substantially extends the set of functional proteins in the individual cells
Separation of methylarginines as basic amino acids is performed in AcOH solutions
At pH 2.45, both the basic groups are protonated, the carboxyl is mostly deprotonated, the overall charge on the amino acid is positive and methylarginines migrate as cations in the cationic direction

Summary

Introduction

N-methylation of the amino acid L-arginine in proteins is a common post-translation modification, which substantially extends the set of functional proteins in the individual cells. Methylation of the N-terminal atoms of the L-arginine side chain is catalysed by a group of enzymes designated as protein arginine methyltransferases (PRMTs), which employ S-adenosylmethionine as a methyl donor [1]. The PRMTs of Class II synthesise MMA in the first phase, but the second methylation leads to the formation of symmetric dimethylarginine (SDMA), see Figure 1. Because of their high basicity, arginine methylated proteins react strongly with nucleic acids and participate in processes such as transcription, translation, etc. The biological function, analysis and identification of arginine methylated proteins is summarised in general reviews [2,3]

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Conclusion