GC-MS and LC-MS/MS pilot studies on the guanidine (NG)-dimethylation in native, asymmetrically and symmetrically NG-dimethylated arginine-vasopressin peptides and proteins in human red blood cells
Graphical abstract
Introduction
Free L-arginine is the substrate of all known nitric oxide synthase (NOS; EC 1.14.13.39) isoforms [1]. The imine group of the terminal guanidine (NG) group of L-arginine is oxidized by NOS to nitric oxide (NO). L-Citrulline is the second product of this reaction. The free amino acids monomethylarginine (MMA, NG-monomethyl-L-arginine), asymmetric dimethylarginine (ADMA, NG,NG-dimethyl-L-arginine) and symmetric dimethylarginine (SDMA, NG,N′G-dimethyl-L-arginine) are endogenous inhibitors of NOS activity [2]. The relative inhibitory potency against the activity of neuronal NOS is MMA > ADMA > SDMA [2]. High concentrations of circulating and low concentrations of urinary ADMA and SDMA are considered cardiovascular risk factors/markers [3]. SDMA is generally considered to be biologically inactive with respect to NOS activity, although SDMA has been reported to inhibit the activity of recombinant neuronal NOS (nNOS) [2]. The cardiovascular risk arising from ADMA is generally attributed to its inhibitory action on endothelial NOS (eNOS). However, the inhibitory potency of ADMA towards eNOS is very weak (e.g., IC50 ≈ 12 µM ADMA) [4]. It is therefore unlikely that the poor inhibitory potency of ADMA and SDMA against eNOS may explain their cardiovascular risks. We hypothesized that these NG-dimethylated Arg analogs may exert additional not yet recognized atherosclerotic effects (reviewed in Ref. [5]). Recently, SDMA, but not ADMA, has been reported to modify the structure of high-density lipoprotein (HDL) and to induce endothelial dysfunction by activating the Toll-like receptor-2 [6], [7], [8]. These newer findings may explain the atherosclerotic effect of SDMA.
Amino acid residues in proteins undergo many different post-translational modifications (PTM) [9], resulting in altered biological functions of those proteins. Two abundant PTM are the citrullination and the methylation of Arg residues (Fig. 1A). The citrullination is catalyzed by the Ca2+-dependent peptidyl-arginine deiminase (PAD; EC 3.5.3.15) [10]. It is assumed that the citrullination is associated with autoimmune diseases, notably rheumatoid arthritis [11]. This is because the newly generated L-citrulline moiety in proteins gives rise to the generation of new autoantibodies. The methylation of certain Arg moieties in proteins is catalyzed by the large family of protein-arginine methyltransferases (PRMT; EC 2.1.1.125), which uses the universal methyl-group donor S-adenosylmethionine (SAM) as the cofactor [12]; PRMT-catalyzed NG-methylation produces proteins that bear MMA, SDMA or ADMA moieties (Fig. 1A). Analogous to citrullination, NG-methylation of proteins may also induce autoimmunity, yet the pathophysiology of this PTM is much less understood. Recent studies indicated that Arg residues in certain transporters and ion channels are NG-dimethylated and that this PTM changes their activity [13], [14], [15].
Identity, biological functions and concentration of NG-methylated Arg proteins are largely unknown. Their proteolysis releases the free amino acids MMA, ADMA and SDMA. They circulate in blood and are distinctly differently metabolized and eliminated by the human body. Thus, SDMA is excreted almost unchanged in the urine. MMA and ADMA are excreted in part unchanged (by about 10% for ADMA) and in part as monomethylamine and dimethylamine (DMA), respectively, upon hydrolysis by dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3.18) [1], [16]. The concentration of the free amino acids ADMA and SDMA in blood and urine are several times higher than that of MMA.
The concentration of asymmetrically NG-dimethylated Arg proteins in human serum is about 5 times lower than that of free ADMA [17]. These ADMA-bearing proteins have not been identified thus far. It is solely known that human serum albumin is essentially free of asymmetrically NG-dimethylated Arg moieties [18]. In serum of healthy elderly subjects, we found an inverse correlation between asymmetric NG-dimethylation and citrullination [17], suggesting potential interaction between these PTM. Human erythrocytes are rich in asymmetrically NG-dimethylated proteins. In proteolysates (6 M HCl, 20 h, 110 °C) of lyzed human erythrocytes, we measured by GC-MS about 15 µM of asymmetrically NG-dimethylated large (>50 kDa) proteins of unknown identity [19]. It is worth mentioning that neither human serum albumin [18] nor human erythrocytic hemoglobin are asymmetrically NG-dimethylated to a considerable degree [20].
The aim of the present study was to contribute to two important aspects of the NG-dimethylation of proteins and peptides: 1) to identify and quantify asymmetrically NG-dimethylated proteins in human red blood cells; and 2) to test the biological activity of NG-dimethylated peptides in comparison to the native non-modified peptides. To reach these goals, we used mass spectrometry-based analytical techniques, i.e., LC-MS/MS and GC-MS approaches, and well-characterized synthetic L-arginine-containing peptides (Fig. 1B). As a model peptide, we chose vasopressin (V), the endogenous anti-diuretic hormone (ADH). The L-arginine-vasopressin (peptide sequence: CYFQNCPRG-NH2) is a nona-peptide and contains a single Arg moiety and two Cys moieties. The biologically and pharmacologically active L-arginine-vasopressin is cyclic (c) due to the Cys-Cys disulfide bridge and the terminal Gly moiety is amidated: cV-Arg-Gly-NH2. In the present work, we used commercially available native arginine-vasopressin (cV-Arg-Gly-NH2) and two custom-made NG-dimethylated arginine-vasopressin analogs: asymmetrically NG-dimethylated arginine-vasopressin (cV-ADMA-Gly-NH2) and the symmetrically NG-dimethylated arginine-vasopressin (cV-SDMA-Gly-NH2). Table 1 and the supplementary Fig. S1 inform of the structures, abbreviations and explanations of the arginine-vasopressin analogs discussed in the present study.
Section snippets
Chemicals and materials
NG,N′G-Di-[2H3]methyl-L-arginine (d6-SDMA, 99% isotopic purity at 2H) and bovine liver catalase were obtained from Sigma-Aldrich (Steinheim, Germany). Sodium azide, acrylamide and dithiothreitol (DTT) were from Merck (Darmstadt, Germany). Arg8-vasopressin (V-Arg) acetate salt (purity, ≥95% by HPLC), angiotensin I and II acetate salt hydrates (declared chemical purity, ≥93% by HPLC), and S-(5′-adenosyl)-L-methionine dihydrochloride (declared chemical purity, ≥75% by HPLC) were purchased from
LC-MS spectra of arginine-vasopressin and analogs
The LC-MS spectrum obtained by positive ESI (+ESI) of the native L-arginine vasopressin form, cV-Arg-Gly-NH2, contained two major mass fragments at m/z 542.7 and 1084.4 in the m/z range 300 – 1600 (Fig. S2A). Very similar LC-MS have been reported for cV-Arg-Gly-NH2 by others, who assigned these ions to the protonated doubly and singly charged molecular cations [M+2H]2+ and [M+H]+, respectively [26], [27], [28]. However, because the molecular mass of the charge-free cV-Arg-Gly-NH2 is 1084.2 (
Discussion
In this work, we performed GC-MS and LC-MS/MS pilot studies on the dimethylation of the guanidine (NG) group of L-arginine in peptides and proteins (Fig. 1). Arginine-vasopressin and custom-made asymmetrically and symmetrically NG-dimethylated vasopressin analogs were used as model peptides. Arginine-vasopressin (V-Arg) is the endogenous nona-peptide Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2. V-Arg contains a single Arg residue on position 8, a disulfide bridge built between two Cys moieties
Conclusion
GC-MS is useful to quantify free ADMA released by classical HCl-catalyzed hydrolysis of synthetic peptides and endogenous NG-dimethylated proteins. LC-MS/MS in the +ESI mode allow the identification of native NG-dimethylated peptides such as cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 upon complete chromatographic separation. The type of NG-dimethylation cannot be determined by CID of [M−H]+/[(M−H)+H]2+, but requires GC-MS analysis of ADMA and SDMA released from the arginine-vasopressin peptides by
CRediT authorship contribution statement
Alexander Bollenbach: Methodology, Investigation, Software, Writing - review & editing, Data curation. Stepan Gambaryan: Methodology, Investigation, Writing - review & editing. Igor Mindukshev: Methodology, Investigation. Andreas Pich: Methodology, Software, Writing - review & editing, Data curation. Dimitrios Tsikas: Conceptualization, Writing - review & editing, Data curation, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
We thank Dr. Ute Gravemann (German Red Cross Blood Service NSTOB, Springe, Germany) for her advice in human platelet aggregation measurements on the aggregometer model PAP-8 and for careful proofreading of our manuscript.
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