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Molecular & Cellular Proteomics 7:459-472, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Quantitative Proteomics Analysis Demonstrates Post-transcriptional Regulation of Embryonic Stem Cell Differentiation to Hematopoiesis^*,S

Andrew J. K. Williamson, Duncan L. Smith, David Blinco, Richard D. Unwin, Stella Pearson, Claire Wilson, Crispin Miller, Lee Lancashire, Georges Lacaud, Valerie Kouskoff and Anthony D. Whetton^,¶

From the Stem Cell and Leukemia Proteomics Laboratory, Faculty of Medical and Human Sciences, University of Manchester, Kinnaird House, Kinnaird Road, Manchester M20 4QL, United Kingdom and Paterson Institute for Cancer Research, Christie Hospital, University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom

Embryonic stem (ES) cells can differentiate in vitro to produce the endothelial and hematopoietic precursor, the hemangioblasts, which are derived from the mesoderm germ layer. Differentiation of Bry^GFP/+ ES cell to hemangioblasts can be followed by the expression of the Bry^GFP/+ and Flk1 genes. Proteomic and transcriptomic changes during this differentiation process were analyzed to identify mechanisms for phenotypic change during early differentiation. Three populations of differentiating Bry^GFP ES cells were obtained by flow cytometric sorting, GFP^–Flk1^– (epiblast), GFP⁺Flk1^– (mesoderm), and GFP⁺Flk1⁺ (hemangioblast). Microarray analyses and relative quantification two-dimensional LCLC-MS/MS on nuclear extracts were performed. We identified and quantified 2389 proteins, 1057 of which were associated to their microarray probe set. These included a variety of low abundance transcription factors, e.g. UTF1, Sox2, Oct4, and E2F4, demonstrating a high level of proteomic penetrance. When paired comparisons of changes in the mRNA and protein expression levels were performed low levels of correlation were found. A strong correlation between isobaric tag-derived relative quantification and Western blot analysis was found for a number of nuclear proteins. Pathway and ontology analysis identified proteins known to be involved in the regulation of stem cell differentiation, and proteins with no described function in early ES cell development were also shown to change markedly at the proteome level only. ES cell development is regulated at the mRNA and protein level.

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