title: Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis.
abstract: The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we provide a detailed method for creating SILAC media suitable for use in hESC experiments, additionally we describe methods for the isolation of membrane fractions and cytosolic and nuclear/membrane fractions.
authors.affiliation: Molecular Endocrinology Laboratory (KMEB), Odense University Hospital, Odense, Denmark. lharkness@health.sdu.dk
authors.forename: Linda
authors.initials: L
authors.lastname: Harkness
authors.forename: Tatyana A
authors.initials: TA
authors.lastname: Prokhorova
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
authors.forename: Blagoy
authors.initials: B
authors.lastname: Blagoev
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*
chemical_list: D000596:Amino Acids