期刊
JOURNAL OF PROTEOME RESEARCH
卷 10, 期 1, 页码 305-319出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr1006203
关键词
epidermal growth factor receptor; tyrosine phosphorylation; mass spectrometry; proteomics; tyrosine kinase inhibitor; erlotinib; lung cancer
资金
- National Functional Genomics Center
- Moffitt Lung SPORE [5P50CA119997]
- US Army Medical Research and Material Command [DAMD17-02-2-0051]
- National Cancer Institute [P30-CA076292]
- Moffitt Foundation
- Florida Department of Health [06BS-02-9614]
- NATIONAL CANCER INSTITUTE [R01CA121182, P50CA119997, P30CA076292] Funding Source: NIH RePORTER
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.
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