The fluorogenic derivatization reagents with a positive charge, 4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benzoxadiazole-4-sulfonylaminoethyltrimethylammonium chloride (TAABD-Cl), are proposed for use in proteomics studies. Following derivatization of protein mixtures with these reagents, a series of standard processes of isolation, digestion, and identification of the proteins were performed utilizing high-performance liquid chromatography-fluorescence detection and tandem mass spectrometry with the probability-based protein identification algorithm. Both DAABD and TAABD derivatives were detected fluorometrically at the femtomole level and showed more than 100-fold improvement in sensitivity compared to the underivatized original compounds with an electrospray ionization ion trap mass spectrometer analysis. The modification of the MASCOT database search system memorized with the fragment information of a DAABD-attached Cys residue allowed the identification of the proteolytic peptide fragments of the derivatized bovine serum albumin (BSA) with an estimated 38% sequence coverage of BSA. Utilizing DAABD-Cl as a derivatization reagent, identification of several proteins was also possible in a soluble extract of Caenorhabditis elegans (10 microg of protein). Consequently, for identification of proteins in the complex matrixes of proteins, DAABD-Cl could be a more appropriate reagent than ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate as reported previously.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now widely used as a tool for proteomic studies. For the sensitive determination of proteins in 2D-PAGE, fluorescence derivatization of primary amino moieties of proteins with cyanine dyes was recently developed. However, precipitation of the proteins could occur if completely derivatized because of the lower solubility of the resultant derivatives owing to the hydrophobicity of the reagents and the loss of the hydrophilic primary amino moieties. Thus, in this paper, a water-soluble and thiol-specific fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, was adopted for the derivatization of proteins in tissues either with and without stimulation. Then, the method follows a separation of the derivatives by liquid chromatography with fluorescence detection, an isolation of only the altered proteins, an enzymatic digestion of the isolated proteins, and an identification of the proteins by liquid chromatography/MS/MS with the database-searching algorithm. By using this method, we identified the altered expressions of five increased proteins (e.g., pancreatic polypeptide) as well as three decreased proteins (e.g., insulin 2) in the islets of Langerhans in Wistar rats 2 days after they were subcutaneously administered with dexamethasone.
Rodents (rat and mouse) have two types of insulin (insulin I and II; each contains a universal chain A and a different composition of each type BI chain or type BII chain). The physiological role for each isomer is not yet clarified because of the lack of an appropriate separative determination method for these isomers. Thus, in this paper, a sensitive and selective HPLC-fluorescence determination method for the isomers was developed, which includes derivatization with a fluorogenic reagent for thiols, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, in the presence of a reducing agent, TCEP, a nonionic surfactant, n-dodecyl beta-D-maltopyranoside, and EDTA. The resultant chain A, BI, and BII derivatives were separated on a reversed-phase column (TSK gel ODS-120T, 250 x 4.6 mm i.d.) with a mobile phase containing 5 mM phosphate buffer (pH 7.0) and were detected at 505 nm with excitation at 380 nm. The detection limits for chain A, BI, and BII derivatives were 2.2, 3.4, and 3.7 fmol on column, respectively. The method was applicable to the determination of rodent insulin in a single islet of Langerhans, and the results indicated its feasibility for the investigation of the pathophysiological roles of the isomers in diabetes in the rodent.
ABSTRACT:The absorption, metabolism, and excretion of imidafenacin [KRP-197/ONO-8025, 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide], a new antimuscarinic drug developed for treatment of overactive bladder, were assessed in six healthy male subjects after a single oral administration of 0.25 mg of [ 14 C]imidafenacin (approximately 46 Ci). The highest radioactivity in the plasma was observed at 1.5 h after administration. The apparent terminal elimination half-life of the total radioactivity was 72 h. Approximately 65.6 and 29.4% of the administered radioactivity were recovered in the urine and feces, respectively, within 192 h after administration. The metabolite profiling by high-performance liquid chromatography-radiodetector and liquid chromatography/tandem mass spectrometry demonstrated that the main component of radioactivity was unchanged imidafenacin in the 2-h plasma. The N-glucuronide conjugate (M-9) was found as the major metabolite and the oxidized form of the 2-methylimidazole moiety (M-2) and the ringcleavage form (M-4) were detected as the minor metabolites in the 2-h plasma, but M-4 was found to be the main component in the 12-h plasma. Unchanged imidafenacin, M-9, M-2, and other oxidized metabolites were excreted in the urine, but the unchanged imidafenacin and M-9 were not found in the feces. Two unique metabolites were found in the urine and feces, which were identified as the interchangeable cis-and trans-isomers of 4,5-dihydrodiol forms of the 2-methylimidazole moiety. These findings indicate that imidafenacin is rapidly and well absorbed (at least 65% of dose recovered in urine) after oral administration, circulates in human plasma as the unchanged form, its glucuronide, and other metabolites, and is then excreted in urine and feces as the oxidized metabolites of 2-methylimidazole moiety.Imidafenacin [KRP-197/ONO-8025, 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide] (Fig. 1) is a newly synthesized antimuscarinic drug developed for treatment of overactive bladder. Acetylcholine is well known for playing a major role in contracting the bladder through activation of muscarinic receptors (Somogyi and de Groat, 1992;Wang et al., 1995;Braverman et al., 1998). Compounds with high affinity for the muscarinic acetylcholine receptor, including propiverine, tolterodine, oxybutynin, darifenacin, and solifenacin, have been used in management of overactive bladder (Chapple et al., 2002;Andersson and Yoshida, 2003;Andersson, 2004;Robinson and Cardozo, 2005). Imidafenacin showed high in vitro affinity for muscarinic receptor subtypes M 1 and M 3 in the functional assay using isolated animal tissues and in the binding assay using recombinant human receptors (Miyachi et al., 1999;Kobayashi et al., 2007a). In addition, imidafenacin inhibited carbachol-induced contraction of isolated guinea pig and human bladder mediated by the M 3 receptor and acetylcholine release from isolated rat and human bladder mediated by the prejunctional M 1 receptor (Murakami et al., 2003;Kobayashi et al., 2007a). A car...
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