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Molecular & Cellular Proteomics 5:57-67, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Identification of Candidate Regulators of Embryonic Stem Cell Differentiation by Comparative Phosphoprotein Affinity Profiling^*,S

Lawrence G. Puente^,,¶, Douglas J. Borris, Jean-François Carrière^,||, John F. Kelly^** and Lynn A. Megeney^,,,

From the Ottawa Health Research Institute, Molecular Medicine Program and the Ontario Genomics Innovation Centre, Ottawa Hospital, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada, the ^** Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada, and the Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic stem cell growth and differentiation correlates with kinase activities, but with the exception of the JAK/STAT3 pathway, the relevant substrates are unknown. To identify candidate phosphoproteins with potential relevance to embryonic stem cell differentiation, a systems biology approach was used. Proteins were purified using phosphoprotein affinity columns, then separated by two-dimensional gel electrophoresis, and detected by silver stain before being identified by tandem mass spectrometry. By comparing preparations from undifferentiated and differentiating mouse embryonic stem cells, a set of proteins was identified that exhibited altered post-translational modifications that correlated with differentiation state. Evidence for altered post-translational modification included altered gel mobility, altered recovery after affinity purification, and direct mass spectra evidence. Affymetrix microarray analysis indicated that gene expression levels of these same proteins had minimal variability over the same differentiation period. Bioinformatic annotations indicated that this set of proteins is enriched with chromatin remodeling, catabolic, and chaperone functions. This set of candidate phosphoprotein regulators of stem cell differentiation includes products of genes previously noted to be enriched in embryonic stem cells at the mRNA expression level as well as proteins not associated previously with stem cell differentiation status.

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