title: A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.
abstract: Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
authors.affiliation: Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Blagoy
authors.initials: B
authors.lastname: Blagoev
authors.forename: Irina
authors.initials: I
authors.lastname: Kratchmarova
authors.forename: Shao-En
authors.initials: SE
authors.lastname: Ong
authors.forename: Mogens
authors.initials: M
authors.lastname: Nielsen
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors