Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection Journal Article


Author(s): Hühner, Jens; Ingles-Prieto, Álvaro; Neusüß, Christian; Lämmerhofer, Michael; Janovjak, Harald
Article Title: Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection
Affiliation IST Austria
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.
Keywords: Optogenetics; Capillary electrophoresis; FAD; FMN; Light-emitting diode-induced fluorescence detection
Journal Title: Electrophoresis
Volume: 36
Issue 4
ISSN: 01730835
Publisher: Wiley-Blackwell  
Date Published: 2015-02-01
Start Page: 518
End Page: 525
Sponsor: Funded by DFG. Grant Number: INST 37/821-1 FUGG
URL:
DOI: 10.1002/elps.201400451
Notes: We thank W. Lindner for advice, M. Spanova (IST Austria) for technical assistance and A. Sievers-Engler (University of Tübingen) for UHPLC-ESI-QTOF-MS measurements. Our work is supported by grants of the European Union Seventh Framework Programme (CIG-303564 to HJ), the Human Frontier Science Program (RGY0084_2012, to HJ), the Struktur- und Innovationsfonds Baden-Württemberg (to ML) and the German Science Funds (DFG no. INST 37/821-1 FUGG) (to ML). AIP is supported by a Dan David scholarship and a Ramon Areces scholarship.
Open access: yes (repository)