title: Mass spectrometry mapping of epidermal growth factor receptor phosphorylation related to oncogenic mutations and tyrosine kinase inhibitor sensitivity.
abstract: The epidermal growth factor receptor (EGFR) plays an important role in cancer by activating downstream signals important in growth and survival. Inhibitors of EGFR are frequently selected as treatment for cancer including lung cancer. We performed an unbiased and comprehensive search for EGFR phosphorylation events related to somatic activating mutations and EGFR inhibitor (erlotinib) sensitivity. EGFR immunoprecipitation combined with high resolution liquid chromatography-mass spectrometry and label free quantitation characterized EGFR phosphorylation. Thirty (30) phosphorylation sites were identified including 12 tyrosine (pY), 12 serine (pS), and 6 threonine (pT). Site-specific phosphorylation was monitored by comparing ion signals from the corresponding unmodified peptide. Phosphorylation sites related to activating mutations in EGFR as well as sensitivity to erlotinib were identified using 31 lung cancer cell lines. We identified three sites (pY1092, pY1110, pY1172) correlated with activating mutations and three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity. Five sites (pT693, pY1092, pY1110, pY1172, and pY1197) were inhibited by erlotinib in concentration-dependent manner. Erlotinib sensitivity was confirmed using liquid chromatography coupled to multiple reaction monitoring (LC-MRM) and quantitative Western blotting. This LC-MS/MS strategy can quantitatively assess site-specific EGFR phosphorylation and can identify relationships between somatic mutations or drug sensitivity and protein phosphorylation.
authors.affiliation: Department of Thoracic Oncology, Molecular Oncology, Biomedical Informatics, Biostatistics, and Proteomics Core Facility, H. Lee Moffitt Cancer Center and Research Institute; Tampa, FL, USA.
authors.forename: Guolin
authors.initials: G
authors.lastname: Zhang
authors.forename: Bin
authors.initials: B
authors.lastname: Fang
authors.forename: Richard Z
authors.initials: RZ
authors.lastname: Liu
authors.forename: Huiyi
authors.initials: H
authors.lastname: Lin
authors.forename: Fumi
authors.initials: F
authors.lastname: Kinose
authors.forename: Yun
authors.initials: Y
authors.lastname: Bai
authors.forename: Umut
authors.initials: U
authors.lastname: Oguz
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Remily-Wood
authors.forename: Jiannong
authors.initials: J
authors.lastname: Li
authors.forename: Soner
authors.initials: S
authors.lastname: Altiok
authors.forename: Steven
authors.initials: S
authors.lastname: Eschrich
authors.forename: John
authors.initials: J
authors.lastname: Koomen
authors.forename: Eric B
authors.initials: EB
authors.lastname: Haura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000069347:Erlotinib Hydrochloride; D047468:Immunoprecipitation; D008175:Lung Neoplasms; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010449:Peptide Mapping / Q000379:methods*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods; D023041:Xenograft Model Antitumor Assays
chemical_list: D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D000069347:Erlotinib Hydrochloride; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases