Existence of low‐molecular‐weight thiols in Caenorhabditis elegans demonstrated by HPLC‐fluorescene detection utilizing 7‐chloro‐N‐[2‐(dimethylamino)ethyl]‐2,1,3‐benzoxadiazole‐4‐sulfonamide

Abstract

A highly sensitive and simple method using HPLC-fluorescence detection with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as a fluorogenic reagent demonstrated the existence of the low-molecular-weight thiols in the extract of Caenorhabditis elegans (C. elegans). The method includes derivatization of the thiols with DAABD-Cl at 40 degrees C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 +/- 15 nm with excitation at 400 +/- 15 nm. The identification of the thiols was made by HPLC-electrospray ionization mass spectrometry (LC-MS) following isolation of the derivatives using HPLC-fluorescence detection. Low-molecular-weight thiols were found to exist in the extract of C. elegans, such as cysteine, cysteinylglycine, gamma-glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the 'glutathione cycle' in C. elegans similar to the mammalian body.

Recent advances in separation and detection methods for thiol compounds in biological samples

“…Imai et al synthesized similar reagents having the benzofurazan structure, i.e., 4-(N-acetylaminosulfonyl)-7-fluoro-2,1, 3-benzoxadiazole (AcABD-F), 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (TCAcABD-F) [121] and 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) [122]. Both the reactivity to the thiol and the fluorescence property of the derivative are almost equivalent to ABD-F. DAABD-Cl was used for the determination of thiols in Caenorhabditis elegans [122].…”

“…Both the reactivity to the thiol and the fluorescence property of the derivative are almost equivalent to ABD-F. DAABD-Cl was used for the determination of thiols in Caenorhabditis elegans [122].…”

Proteomics of Caenorhabditis elegans over‐expressing human α‐synuclein analyzed by fluorogenic derivatization–liquid chromatography/tandem mass spectrometry: identification of actin and several ribosomal proteins as negative markers at early Parkinson's disease stages

“…Previously, we have developed a novel method of proteome analysis, called fluorogenic derivatization liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS), which includes the first fluorogenic derivatization (FD) of protein mixtures, isolation of the target proteins with high-performance liquid chromatography (HPLC) separation, followed by enzymatic digestion and identification of the derivatized proteins by LC-tandem mass spectrometry (MS/MS) with the probability-based protein identification algorithm (Asamoto et al, 2007;Masuda et al, 2004 and. This method has high selectivity, sensitivity and repeatability utilizing DAABD-Cl as a fluorogenic reagent and HPLC as a separation technique.…”

FD-LC-MS/MS Method for Determining Protein Expression and Elucidating Biochemical Events in Tissues and Cells

“…24,25) 5) In the post-genome era, a sensitive quantitative method for differential profiling analyses of clinical proteomics is required to understand the disease progress. Therefore, as a preliminary step, we adopted an FD-LC-MS/ MS method using DAABD-Cl to clarify disease-related liver proteins in hepatocarcinogenesis in transgenic (Tg) and non-transgenic (NTg) mice at three different developmental stages.…”