As the functions of proteins are associated with their cellular localization, the comprehensive sub-cellular proteome knowledge of human embryonic stem cells (hESCs) is indispensable for ensuring a therapeutic effect. Here, we have utilized a sub-cellular proteomics approach to analyze the localization of proteins in the nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomes. Out of 2002 reproducibly identified proteins, we detected 762 proteins in a single organelle whereas 160 proteins were found in all sub-cellular fractions. We verified the localization of identified proteins through databases and discussed the consistency of the obtained results. With regards to the ambiguity in the definition of a membrane protein, we tried to clearly define the plasma membrane, peripheral membrane and membrane proteins by annotation of these proteins in databases, along with predictions of transmembrane helices. Among ten enriched signaling pathways highlighted in our results, non-canonical Wnt signaling were analyzed in greater detail. The functions of three novel hESC membrane proteins (ERBB4, GGT1 and ZDHHC13) have been assessed in terms of pluripotency. Our report is the most comprehensive for organellar proteomics of hESCs. Significance Mass spectrometric identification of proteins using a TripleTOF 5600 from nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomal fractions highlighted the significance of the non-canonical Wnt signaling in human embryonic stem cells.
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