title: Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood.
abstract: Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQ(TM)) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia.
authors.affiliation: Cell Signaling Laboratory, Department of Biomedical Science (DIBINEM), University of Bologna, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Piazzi
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Williamson
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.
authors.forename: Chia-Fang
authors.initials: CF
authors.lastname: Lee
authors.affiliation: Stem Cell Research Group, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK.
authors.forename: Stella
authors.initials: S
authors.lastname: Pearson
authors.affiliation: Stem Cell Biology Group Paterson Institute for Cancer Research, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK.
authors.forename: Georges
authors.initials: G
authors.lastname: Lacaud
authors.affiliation: Stem Cell Research Group, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK.
authors.forename: Valerie
authors.initials: V
authors.lastname: Kouskoff
authors.affiliation: Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: McCubrey
authors.affiliation: Cell Signaling Laboratory, Department of Biomedical Science (DIBINEM), University of Bologna, Italy.
authors.forename: Lucio
authors.initials: L
authors.lastname: Cocco
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK.
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Whetton
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D002853:Chromatography, Liquid; D030562:Databases, Protein; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005326:Fetal Proteins / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D055018:Hemangioblasts / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism; D000098908:Brachyury Protein
chemical_list: D005326:Fetal Proteins; D010750:Phosphoproteins; D020825:T-Box Domain Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2; D000098908:Brachyury Protein
keywords: hemangioblast; iTRAQ; nucleus; phosphoproteomic