GC-MS and LC-MS/MS pilot studies on the guanidine (N)-dimethylation in native, asymmetrically and symmetrically N-dimethylated arginine-vasopressin peptides and proteins in human red blood cells.

Protein-arginine methyltransferases catalyze the methylation of the guanidine (N) group of proteinic L-arginine (Arg) to produce monomethyl and dimethylarginine proteins. Their proteolysis releases the free amino acids monomethylarginine (MMA), symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA), respectively. MMA, SDMA and ADMA are inhibitors of the nitric oxide synthase (NOS) activity. High circulating and low urinary concentrations of ADMA and SDMA are considered risk factors in the cardiovascular and renal systems, mainly due to their inhibitory action on NOS activity. Identity, biological activity and concentration of N-methylated proteins are largely unknown. The present study addressed these issues by using GC-MS and LC-MS/MS approaches. GC-MS was used to quantify free ADMA released by classical HCl-catalyzed hydrolysis of three synthetic Arg-vasopressin (V) peptides and of unknown endogenous N-dimethylated proteins. The cyclic (c) disulfide forms of Arg-vasopressin analogs, i.e., Arg-vasopressin (cV-Arg-Gly-NH), asymmetrically N-dimethylated vasopressin (cV-ADMA-Gly-NH) and symmetrically N-dimethylated vasopressin (cV-SDMA-Gly-NH) were used as model peptides in quantitative GC-MS analyses of ADMA, SDMA and other expected amino acids from the hydrolyzed Arg-vasopressin analogs. cV-ADMA-Gly-NH and cV-SDMA-Gly-NH were discriminated from cV-Arg-Gly-NH by LC-MS and LC-MS/MS, yet they were indistinguishable from each other. The same applies to the respective open (o) reduced and di-S-acetamide forms of oV-ADMA-Gly-NH, oV-SDMA-Gly-NH and oV-Arg-Gly-NH. Our LC-MS and LC-MS/MS studies suggest that the Arg-vasopressin analogs form [(M-H)] and [(M-H)+H] in the positive ESI mode and undergo in part conversion of their terminal Gly-NH (NH, 16 Da) group to Gly-OH (OH, 17 Da). The product ion mass spectra of the di-S-acetamide forms are complex and contain several intense mass fragments differing by 1 Da. cV-ADMA-Gly-NH and cV-SDMA-Gly-NH induced platelet aggregation in platelet-rich human plasma with moderately different initial velocity and maximal aggregation rates compared to cV-Arg-Gly-NH. Previous studies showed that human red blood cells are rich in large (>50 kDa) ADMA-containing proteins of unknown identity. Our LC-MS/MS proteomic study identified several membrane and cytosolic erythrocytic N-dimethylated proteins, including spectrin-α (280 kDa), spectrin-β (247 kDa) and protein 4.1 (80 kDa). Being responsible for the stability of the erythrocyte membrane, the newly identified main targets for N-dimethylation in human erythrocytes should be given a closer look in erythrocytic diseases like hereditary spherocytosis.