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Molecular & Cellular Proteomics 8:959-970, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Quantitative Comparison of the Membrane Proteomes of Self-renewing and Differentiating Human Embryonic Stem Cells^*,S

Tatyana A. Prokhorova^,,¶, Kristoffer T. G. Rigbolt^,¶, Pia T. Johansen^,¶, Jeanette Henningsen^,||, Irina Kratchmarova^,**, Moustapha Kassem^, and Blagoy Blagoev^,

From the Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark, Molecular Endocrinology Lab, Department of Endocrinology, Odense University Hospital and Medical Biotechnology Center, Winslowsparken 25, DK-5000 Odense C, Denmark, and ^|| Centre of Inflammation and Metabolism, University of Copenhagen, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.

To whom correspondence may be addressed: Tel.: 45-6550-2366; Fax: 45-6593-3018; E-mail: bab{at}bmb.sdu.dk

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