Optimization of SILAC in human embryonic stem cells

We present a fully defined culture system (adapted Essential8 (E8) medium in combination with vitronectin) for human embryonic stem cells (hESC) that can be used for Stable Isotopic Labeling of Amino aCids (SILAC) purposes. Although a complete incorporation of the labels was observed after 4 days in culture, more than 50 % of all mass spectrometry (MS) precursors displayed a conversion of L-arginine (R) to L-proline (P) or L-glumatate (E) of 40 %. To reduce this arginine conversion, E8 medium was modified by adding (1) L-proline, (2) L-ornithine, (3) Nω-hydroxy-nor-L-arginine (Nor-NOHA) acetate or by (4) lowering the arginine concentration. Inhibition of arginine conversion was best obtained by adding 5mM L-ornithine, followed by 3.5 mM L-proline and by lowering arginine concentration to 99.5 µM. No major changes in the proteome (HDMSE data), pluripotency and cell amount could be observed for these adapted Essential8TM media, although sudden cell death was sometimes observed with the use of 99.5 µM L-arginine. In conclusion, we suggest using 5 mM L-ornithine to reduce arginine conversion.