Introduction

Asymmetric dimethylarginine (ADMA) is a naturally occurring amino acid that was originally identified in plasma and urine (1,2,3). ADMA is a competitive inhibitor of all nitric oxide synthase (NOS) isoforms and thus modulates the biological effects of NO, especially in the cardiovascular system (1,4). Several studies have suggested that ADMA plasma levels constitute a marker of endothelial dysfunction and cardiovascular disease (5,6,7).

Three main forms of methylarginine have been identified in eukaryotes as N^G-monomethylarginine (L-NMMA), N^GN^G (asymmetric) dimethylarginine (ADMA), and N^GN^′G (symmetric) dimethylarginine (SDMA), all characterized by methylation of one or both guanidine nitrogen atoms of arginine. Methylarginines are generated by the action of protein arginine methyltransferases (PRMTs), which utilize S-adenosylmethionine (AdoMet) as a methyl donor (8,9). Type I PRMTs catalyze ADMA formation in select target proteins, whereas type II PRMTs catalyze SDMA formation through methylation of both guanidino nitrogens. In addition, all PRMTs can also catalyze monomethylation, thereby leading to the formation of L-NMMA (8,9).

Up to now, the biological impact of protein arginine methylation remains to be fully elucidated, but this process is reported to be involved in the regulation of RNA binding, control of transcription, DNA repair, protein localization, protein-protein interaction, signal transduction, and recycling of receptors (9). Large-scale proteomic approaches have unraveled a potentially broad range of substrate proteins for PRMT methylation (10,11), suggesting a significant role for arginine methylation in cellular processes. Moreover, increased methylation of cellular proteins by PRMTs may significantly contribute to the level of free methylarginines via protein degradation (12,13), as direct methylation of free arginines has thus far not be demonstrated.

Sensitive and accurate quantification methods for L-arginine (L-Arg), L-NMMA, ADMA, and SDMA involve ion-exchange extraction from plasma, urine, or cerebrospinal fluid, followed by high-performance liquid chromatography (HPLC) separation with fluorescence detection (14,15,16,17,18,19,20), although capillary electrophoresis (1,21) and liquid chromatography/mass spectrometry (LC/MS) have also been employed (22,23). None of the reported methods, however, have ever been used for the quantification of the degree of protein arginine methylation in the proteome of biological samples. In the present study, an approach using protein precipitation and hydrolysis combined with HPLC separation and fluorescence detection for the simultaneous quantification of L-Arg, L-NMMA, ADMA, and SDMA in cellular and tissue proteins was developed. This method allowed us to calculate the degree of methylation of protein-incorporated and free arginines, demonstrating dynamic changes in arginine methylation in cellular lysates of proteasome-inhibited A549 cells in vitro, as well as differential arginine methylation patterns comparing mouse heart with kidney tissue extracts in vivo.

Materials and Methods

Materials and HPLC System

L-Arg, L-NMMA, L-homoarginine, ADMA, SDMA, and myelin basic protein (MBP) from bovine brain were purchased from Sigma (St. Louis, MO, USA), while acetonitrile, methanol (both LiChrosolv), and concentrated (25%, v/v) ammonia were obtained from Merck (Darmstadt, Germany). Ortho-phthaldialdehyde (OPA) and borate buffer were purchased from Grom (Rottenburg-Hailfingen, Germany). Oasis® MCX cation-exchange solid phase extraction (SPE) columns (30 mg, 1 mL) were supplied by Waters (Eschborn, Germany). All other chemicals were of analytical grade. Separation of amino acids was performed on a HPLC system consisting of an WISP 710B auto sampler, a model 6000 pump, a model 470 fluorescence detector (Waters) and a data acquisition system (Maxima 820, version 3.31; Millipore, Schwalbach, Germany).

Sample Preparation and Protein Hydrolysis

Mouse hearts and kidneys were surgically excised after thoracotomy, immediately homogenized in liquid nitrogen followed by addition of ice-cold cell lysis buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton® X-100, 2 mM Na₃VO₄]. Homogenized tissue was incubated for 1 h on ice and centrifuged for 15 min at 16,000× g. The resulting supernatant was stored at –20°C. Proteins were then precipitated by mixing 100 µL tissue/cell extracts with an equal volume of 20% (v/v) trichloroacetic acid for 20 min. After centrifugation at 16,000× g for 12 min, the supernatants were removed, and the protein pellets were washed with 100 µL ice-cold acetone for 60 min at –20°C. The suspension was centrifuged at 16,000× g for 12 min, and the resulting protein pellet was dissolved in 100 µL distilled water. Precipitation of MBP (100 µL of a 0.25 µg/µL solution) was similarly performed prior to protein hydrolysis, SPE, derivatization, and chromatographic separation. Protein concentrations of crude extracts and precipitated proteins were determined by the Bio-Rad Protein Assay Dye Reagent Concentrate using a SmartSpec™ 3000 spectrophotometer (Bio-Rad Laboratories, Hercules, California, USA). Prior to protein hydrolysis, 20 µL of each sample were combined with 10 µL L-homoarginine (4 pmol/µL) as an internal standard. Total hydrolysis of precipitated protein fractions was achieved by gas-phase hydrolysis with 6 M HCl (constant boiling, sequencing grade; Pierce, Bonn, Germany) at 110°C for 16 h. Samples were dried by use of a vacuum centrifuge and stored at –20°C until further analyzed.