title: Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells.
abstract: We present a fully defined culture system (adapted Essential8TM[E8TM] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8TMmedium was modified by adding (1) l-proline, (2) l-ornithine, (3) Nω-hydroxy-nor-l-arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l-ornithine, followed by 3.5 mM l-proline and by lowering the arginine concentration in the medium to 99.5 μM. No major changes in pluripotency and cell amount could be observed for the adapted E8TMmedia with ornithine and proline. However, our subsequent ion mobility assisted data-independent acquisition (high-definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion.
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Ellen
authors.initials: E
authors.lastname: Scheerlinck
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Katleen
authors.initials: K
authors.lastname: Van Steendam
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Simon
authors.initials: S
authors.lastname: Daled
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Govaert
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Liesbeth
authors.initials: L
authors.lastname: Vossaert
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Paulien
authors.initials: P
authors.lastname: Meert
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Filip
authors.initials: F
authors.lastname: Van Nieuwerburgh
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
authors.affiliation: Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Luc
authors.initials: L
authors.lastname: Peelman
authors.affiliation: Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Petra
authors.initials: P
authors.lastname: De Sutter
authors.affiliation: Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Björn
authors.initials: B
authors.lastname: Heindryckx
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Dhaenens
authors.affiliation: ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium. dieter.deforce@ugent.be.
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
mesh_terms: D001120:Arginine / Q000032:analysis / Q000378:metabolism*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism*; D000066449:Human Embryonic Stem Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D050814:Octamer Transcription Factor-3 / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D003470:Culture Media; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D020543:Proteome; D001120:Arginine
keywords: Arginine conversion; Cell culture; SILAC; Technology; hESC