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Title page for ETD etd-01242012-122721


Type of Document Dissertation
Author Myers, Matthew V
URN etd-01242012-122721
Title Proteomic Signatures of Epidermal Growth Factor Receptor Signaling
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Daniel C. Liebler Committee Chair
Carlos Arteaga Committee Member
Graham Carpenter Committee Member
Jennifer Pietenpol Committee Member
Robert Coffey Committee Member
Keywords
  • mass spectrometry
  • quantitation
  • proteomics
  • biochemistry
  • EGFR
Date of Defense 2011-12-19
Availability unrestricted
Abstract
Analysis of cellular signaling networks typically involves targeted

measurements of phosphorylated protein intermediates. However,

phosphoproteomic analyses usually require affinity enrichment of

phosphopeptides and can be complicated by artifactual changes in

phosphorylation caused by uncontrolled preanalytical variables, particularly in

the analysis of tissue specimens. I hypothesized that changes in protein

expression, which are more stable and easily analyzed, could reflect network

stimulation and inhibition. This approach was employed to analyze stimulation

and inhibition of the epidermal growth factor receptor (EGFR) by EGF and

selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating

A431 cells, EGF-stimulated cells and cells co-treated with the EGFR inhibitors

cetuximab or gefitinib identified groups of differentially expressed proteins.

Comparisons of these protein groups identified 13 proteins whose EGF-induced

expression changes were reversed by both EGFR inhibitors. Targeted multiple-

reaction-monitoring (MRM) analysis verified differential expression of 12 of

these proteins, which comprise a candidate EGFR inhibition signature. I then

tested these 12 proteins by MRM analysis in 3 other models: 1) a comparison of

DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in

formalin-fixed, paraffin-embedded (FFPE) mouse xenograft DiFi and HCT116

tumors, and 3) in tissue biopsies from a patient with the gastric

hyperproliferative disorder Ménétrier’s disease, who was treated with cetuximab.

Of the proteins in the candidate signature, a core group, including c-Jun,

jagged-1, and claudin 4 were decreased by EGFR inhibitors in all three models.

Although the goal of these studies was not to validate a clinically-useful EGFR

inhibition signature, the results confirm the hypothesis and outline a

prototypical approach to derive and test protein expression signatures for drug

action on signaling networks.

A secondary goal of this research was to apply a new method to quantify

protein modification changes to EGFR using internal reference peptides (IRP).

The major focus of this work was to assess the performance of this newly

developed MS-based quantitation method to detect phosphorylation changes on

EGFR by comparing the performance characteristics to stable isotope dilution

(SID) methods. Initial studies are presented along with suggestions for future

studies using overall findings in this dissertation.

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