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Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach

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  • Elisabetta Boeri Erba
  • Rune Matthiesen
  • Jakob Bunkenborg
  • Waltraud X Schulze
  • Paola Di Stefano
  • Sara Cabodi
  • Guido Tarone
  • Paola Defilippi
  • Ole N Jensen
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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.
Original languageEnglish
JournalJournal of Proteome Research
Volume6
Issue number7
Pages (from-to)2768-85
Number of pages18
ISSN1535-3893
DOIs
Publication statusPublished - 2007
Externally publishedYes

    Research areas

  • Amino Acid Sequence, Cell Adhesion, Enzyme Inhibitors, HeLa Cells, Humans, Isotope Labeling, Mass Spectrometry, Molecular Sequence Data, Phosphopeptides, Phosphorylation, Phosphotyrosine, Protein Tyrosine Phosphatases, Receptor, Epidermal Growth Factor, Tyrosine, Vanadates

ID: 42360604