An improved method for proteomics studies inC. elegans by fluorogenic derivatization, HPLC isolation, enzymatic digestion and liquid chromatography-tandem mass spectrometric identification

Abstract

An improved method for proteomics studies, which includes the fluorogenic derivertization of protein mixtures with 7-chloro-4-(dimethylaminoethylaminosulfonyl)-2,1,3-benzoxadiazole (DAABD-Cl), followed by HPLC isolation, enzymatic digestion and identification of the derivatized proteins by HPLC-electrospray ionization (ESI)-MS/MS with the probability-based protein identification algorithm, identified 103 proteins in the soluble extract (10 microg protein) of Caenorhabditis elegans.

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

“…The extraction procedure of the proteins from the α-synWT and the control C. elegans was performed according to a previous report (Masuda et al, 2005). For the FD reaction, a 5.0 μL aliquot (75 μg of protein) of the extracted proteins was mixed with a 30 μL mixture of 0.83 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 3.3 mM ethylenediamine-N,N,N′,N′-tetraacetic acid sodium salt (Na 2 EDTA) and 16.6 mM CHAPS in the pH 8.7 buffer solution, 12.5 μL of the buffer solution and 2.5 μL of 140 mM DAABD-Cl in acetonitrile.…”

Recent progress in the development of derivatization reagents having a benzofurazan structure

“…This method, called fluorogenic derivatization liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS), is basically different from the 2D-PAGE and ICAT method, as mentioned above. The FD-LC-MS/MS method permitted the identification of more than 100 proteins in the soluble extract of Caenorhabditis elegans (C. elegans; 10 μg protein; Masuda et al, 2005). The method was also applied to the soluble extract of C. elegans and of mouse liver, and elucidated for the first time the subtle changes of proteins at early-stage Parkinson's disease and at the three progression stages of hepatocarcinogenesis , respectively.…”

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

“…However, many interfering peaks appeared to obstruct the peak identity of lowmolecular-weight thiols such as Cys, HCys and GSH. Therefore, re-investigation was performed of the previous preparation procedures of the derivatives (Masuda et al, 2004) and the separation conditions (Masuda et al, 2005) for the thiols (Cys, HCys and GSH with the addition of Cys-Gly, cysteamine, NAC and oxytocin).…”

“…Utilizing these reagents, we have proposed a novel method for proteomics studies, which includes derivatization of proteins with these reagents followed by isolation of the derivatized proteins using HPLC-fluorescence detection (FD), digestion of the derivatized proteins and identification of the proteins utilizing electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the probability-based protein identification algorithm (FD-LC-MS/MS method; Toriumi and Imai, 2003;Masuda et al, 2004). In particular, DAABD-Cl was suited to the method, and allowed for the identification of more than 100 proteins in a soluble extract of Caenorhabditis elegans (C. elegans: strain Bristol N2) (Masuda et al, 2005). Consequently, the FD-LC-MS/MS method utilizing DAABD-Cl offers determination and profile analysis of the proteins in the biological samples, and is now being applied to proteomics studies in animal pathogenesis.…”