title: Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.
abstract: Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.
authors.affiliation: IST Austria (Institute of Science and Technology Austria), Klosterneuburg, Austria; Faculty of Chemistry, Aalen University, Aalen, Germany; Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Hühner
authors.forename: Álvaro
authors.initials: Á
authors.lastname: Ingles-Prieto
authors.forename: Christian
authors.initials: C
authors.lastname: Neusüß
authors.forename: Michael
authors.initials: M
authors.lastname: Lämmerhofer
authors.forename: Harald
authors.initials: H
authors.lastname: Janovjak
mesh_terms: D000818:Animals; D016466:CHO Cells; D002138:Calibration; D002460:Cell Line; D002478:Cells, Cultured; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005486:Flavin Mononucleotide / Q000032:analysis*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis*; D057809:HEK293 Cells; D006801:Humans; D054023:Lasers, Semiconductor; D008322:Mammals; D051379:Mice; D041681:NIH 3T3 Cells; D015203:Reproducibility of Results; D012256:Riboflavin / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D012256:Riboflavin
keywords: Capillary electrophoresis; FAD; FMN; Light-emitting diode-induced fluorescence detection; Optogenetics