Background and Objectives: Metabolic labeling with stable isotopes remains a prominent technique for comparative quantitative proteomics and stable isotope labeling with amino acids in cell culture (SILAC) is the most prominent approach used. However, despite its power the approach traditionally is limited if applied to complex tissue culture regiments as those required for human embryonic stem cells (hESC). Classic hESC culture is based on use of mouse embryonic fibroblasts (MEFs) for conditioning the cell culture medium or as a feeder layer. As a result the possibility of xenogeneic contaminants, contribution of unlabeled amino acids, inter-laboratory variability of MEFs and the trick complexity of the culture system are all concerns when using SILAC and beyond. Methods: We applied high accuracy LC-MS/MS analyses to evaluate the SILAC labeling efficiency of the hESCs cultured in the new SILAC- hESCs culture system. Results: The analysis yielded over 15,00 distinct hESC proteins with more than 99% accuracy of identification as estimated by reverse database searching. The efficiency of labeling was estimated to be higher than 99% for lysine and arginine; moreover, SILAC-labeled hESCs maintained undifferentiated self-renewal status. Conclusions: Here we use an enhanced feeder-free SILAC culture system based on a customized chemically defined SILAC-medium and a modified culture protocol to overcome these limitations and achieve reproducible labeling in a process easily scaled to proteomic protein requirements. The protocol is expected to greatly enhance the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESC differentiation and self-renewal.


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