title: Identification of cytoplasmic and membrane-associated complexes in human embryonic stem cells using blue native PAGE.
abstract: Human embryonic stem cells (hESCs) have great potential for use in developmental biology studies, functional genomics applications, drug screening, and regenerative medicine. A detailed understanding of the molecular mechanisms that are responsible for maintaining the undifferentiated and pluripotent nature of hESCs is essential for their effective therapeutic application. It has become evident that many complex cellular processes are carried out by assemblies of protein molecules (protein complexes). Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to separate protein complexes from whole cell lysates. Using BN-PAGE, we resolved cytoplasmic and membrane-associated complexes from hESCs and characterised their composition, stoichiometry, and dynamics by denaturing SDS-PAGE. The reliability of the fractionation was examined by western blot analysis of membrane and cytosolic markers. MALDI TOF/TOF mass spectrometry identified 119 cytosolic and 69 membrane proteins from the BN-PAGE proteome maps. Potential protein complexes were validated by computational prediction of possible protein-protein interactions using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Based on BN-PAGE gels and validation by databases, 82 heteromultimeric and 47 homomultimeric protein complexes have been found in hESCs. Resolving some of the protein complexes provided insight into the function of previously uncharacterised complexes in hESCs.
authors: Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors: Faezeh
authors: F
authors: Shekari
authors: Adele
authors: A
authors: Taei
authors: Tai-Long
authors: TL
authors: Pan
authors: Pei-Wen
authors: PW
authors: Wang
authors: Hossein
authors: H
authors: Baharvand
authors: Ghasem Hosseini
authors: GH
authors: Salekdeh
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D011485:Protein Binding
chemical_list: D008565:Membrane Proteins