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Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells
Author(s) -
Scheerlinck Ellen,
Steendam Katleen,
Daled Simon,
Govaert Elisabeth,
Vossaert Liesbeth,
Meert Paulien,
Nieuwerburgh Filip,
Soom Ann,
Peelman Luc,
Sutter Petra,
Heindryckx Björn,
Dhaenens Maarten,
Deforce Dieter
Publication year - 2016
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201600174
Subject(s) - stable isotope labeling by amino acids in cell culture , arginine , ornithine , embryonic stem cell , arginase , proline , biochemistry , microbiology and biotechnology , chemistry , amino acid , proteome , cell culture , biology , proteomics , genetics , gene
We present a fully defined culture system (adapted Essential8 TM [E8 TM ] 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, E8 TM medium was modified by adding (1) l ‐proline, (2) l ‐ornithine, (3) N ω ‐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 E8 TM 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.