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Sci. Signal., 15 March 2011
Vol. 4, Issue 164, p. rs3
[DOI: 10.1126/scisignal.2001570]

RESEARCH RESOURCES

System-Wide Temporal Characterization of the Proteome and Phosphoproteome of Human Embryonic Stem Cell Differentiation

Kristoffer T. G. Rigbolt1*, Tatyana A. Prokhorova1*, Vyacheslav Akimov1, Jeanette Henningsen1,2, Pia T. Johansen3, Irina Kratchmarova1, Moustapha Kassem3,4, Matthias Mann5,6, Jesper V. Olsen6{dagger}, and Blagoy Blagoev1{dagger}

1 Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
2 Centre of Inflammation and Metabolism, University of Copenhagen, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
3 Molecular Endocrinology Laboratory, Department of Endocrinology, Odense University Hospital and Medical Biotechnology Center, Winslowsparken 25, DK-5000 Odense, Denmark.
4 Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, 11461 Riyadh, Kingdom of Saudi Arabia.
5 Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
6 Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark.

* These authors contributed equally to this work.

Abstract: To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.

{dagger} To whom correspondence should be addressed. E-mail: bab{at}bmb.sdu.dk (B.B.); jesper.olsen{at}cpr.ku.dk (J.V.O.)

Citation: K. T. G. Rigbolt, T. A. Prokhorova, V. Akimov, J. Henningsen, P. T. Johansen, I. Kratchmarova, M. Kassem, M. Mann, J. V. Olsen, B. Blagoev, System-Wide Temporal Characterization of the Proteome and Phosphoproteome of Human Embryonic Stem Cell Differentiation. Sci. Signal. 4, rs3 (2011).

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