Development and validation of an improved diced electrophoresis gel assay cutter-plate system for enzymomics studies

Publication date: Available online 19 June 2018Source: Biochimica et Biophysica Acta (BBA) - Proteins and ProteomicsAuthor(s): Toru Komatsu, Masahiro Shimoda, Yukiko Kawamura, Yasuteru Urano, Tetsuo NaganoAbstractDiced electrophoresis gel (DEG) assay is a methodology to identify enzymes with a specified activity in complex cell or tissue lysates by means of two-dimensional separation using isoelectric focusing and native PAGE, followed by dicing of the gel into small pieces that are assayed separately, and digestion and peptide fingerprinting to identify the protein(s) of interest in positive wells. The existing hand-made system has some disadvantages, and here we describe the development and validation of an improved cutter-plate system that enables simple, reliable and reproducible DEG assay in a 384-well plate-based format with signal readout using fluorometric or LC-MS-based reaction monitoring. To illustrate the usefulness of this system, we describe its application to profile esterase activities in ovarian adenocarcinoma SKOV3 cell lysate and mouse liver lysate that activate a fluorogenic substrate, fluorescein dibutyrate (FDBu), as well as esterase activities in mouse liver lysate that activate S-bromobenzylglutathione dicyclopentyl ester (BBGDC), a prodrug of anti-tumor agent S-bromobenzylglutathione. The activity spot patterns detected for FDBu and BBGDC were completely different, indicating that different metabolic systems are involved in hydrolysis of these substra...
Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - Category: Biochemistry Source Type: research