Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells |
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| Authors: | Ellen Scheerlinck Katleen Van Steendam Simon Daled Elisabeth Govaert Liesbeth Vossaert Paulien Meert Filip Van Nieuwerburgh Ann Van Soom Luc Peelman Petra De Sutter Björn Heindryckx Dieter Deforce |
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| Affiliation: | 1. ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Belgium;2. Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium;3. Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Belgium;4. Ghent Fertility and Stem Cell Team (G‐FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium;5. ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, BelgiumBoth the authors contributed equally to this work. |
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| 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, E8TM medium 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 E8TM media 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. |
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| Keywords: | Arginine conversion Cell culture hESC SILAC Technology |
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