title: Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach.
abstract: Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium pervanadate to boost the abundance of phosphorylation of the EGF receptor. Nine phosphorylation sites (pT669, pS967, pS1002, pY845, pY974, pY1045, pY1086, pY1148, and pY1173) of EGF receptor were quantified from EGF-stimulated cells in suspension and adherent conditions. Our data sets revealed that EGF stimulation of adherent cells induced higher levels of tyrosine phosphorylation relative to EGF stimulation of suspended cells. In contrast, EGF stimulation of adherent cells induced lower levels of serine and threonine phosphorylation relative to EGF stimulation of suspended cells. These findings are consistent with the hypothesis that cellular adhesion modulates phosphorylation of plasma membrane receptor tyrosine kinases relevant for EGF-induced signal transduction processes. Furthermore, our results suggest that strong phosphatase inhibitors should be used to generate reference datasets in comparative phosphoproteomics experiments.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Boeri Erba
authors.forename: Rune
authors.initials: R
authors.lastname: Matthiesen
authors.forename: Jakob
authors.initials: J
authors.lastname: Bunkenborg
authors.forename: Waltraud X
authors.initials: WX
authors.lastname: Schulze
authors.forename: Paola
authors.initials: P
authors.lastname: Di Stefano
authors.forename: Sara
authors.initials: S
authors.lastname: Cabodi
authors.forename: Guido
authors.initials: G
authors.lastname: Tarone
authors.forename: Paola
authors.initials: P
authors.lastname: Defilippi
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D000595:Amino Acid Sequence; D002448:Cell Adhesion; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000032:analysis*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D010748:Phosphopeptides; C076715:pervanadate; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases