title: On-chip enzymatic assay for chloramphenicol acetyltransferase using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
abstract: Herein, we report a chloramphenicol (CAP) acetyltransferase (CAT) activity assay based on self-assembled monolayers on gold as an alternative to conventional CAT reporter gene assay systems, which sometimes require toxic materials and complicated steps that limit their use. A CAP derivative presented on a monolayer was converted to the acetylated CAP by CAT in the presence of acetyl-CoA. The conversion was directly monitored by observing the molecular weight changes in CAP using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CAT activity was determined under various reaction conditions by changing reaction times, CAT and acetyl-CoA concentrations. As a practical application, we identified gene expression in bacteria that were transformed with pCAT plasmid DNA. Our strategy can provide a simple and rapid assay that eliminates some commonly used but potentially detrimental steps in enzymatic assays, such as radioactive labeling and complicated separation and purification of analytes prior to detection.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Republic of Korea; Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Inseong
authors.initials: I
authors.lastname: Choi
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Republic of Korea.
authors.forename: Dong-Eun
authors.initials: DE
authors.lastname: Kim
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Republic of Korea; Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Joong-Hoon
authors.initials: JH
authors.lastname: Ahn
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Republic of Korea; Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea. Electronic address: wsyeo@konkuk.ac.kr.
authors.forename: Woon-Seok
authors.initials: WS
authors.lastname: Yeo
mesh_terms: D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism*; D007700:Kinetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D015500:Chloramphenicol O-Acetyltransferase
keywords: Chloramphenicol acetyltransferase; Mass spectrometry; Monolayers; Reporter gene assay