iConcept Press Logo
Email Password Remember me
Protein Purification: Principles and Trends
Title
Protein Purification: Principles and Trends
Editor
iConcept Press
Price
USD$89.00
ISBN
978-1-922227-40-9
Clicks
20057

Chapter 1

Protein Purification: Principles and Trends

Discovery of Biomarkers Mediating EGFR Tyrosine Kinase Inhibitor Resistance in Non-small Cell Lung Cancer: A Proteomics Approach

by Gregory Botting, Ichwaku Rastogi, Gagan Chhabra, Jason T Fong, Ryan J. Jacobs, Caleb Shearrow, David N. Moravec, Joseph Devito, Kymberly Harrington, Ryan D. Bomgarden, John C. Rogers and Neelu Puri

Viewed: 1943

Abstract

EGFR is a receptor tyrosine kinase (RTK) that play a significant role in the tumorgenicity of non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitor (TKI) erlotinib specific for EGFR, have been developed to inhibit progression of NSCLC. Currently EGFR TKIs have been approved for the treatment of NSCLC; however, resistance to EGFR TKIs is a well documented modality for recurrence of NSCLC. c-Met amplification accounts for 20% of acquired resistance and a secondary “gatekeeper” mutation, T790M, accounts for 50% of all acquired resistance to EGFR TKIs. To determine proteins involved in EGFR TKI resistance mechanism, we initially developed H358 lung cancer cells to be resistant to high concentrations of erlotinib (erlotinib IC50 concentrations 4-5 times higher than parental cells). By analyzing altered expression of proteins in EGFR TKI resistant cells, we may gain a better understanding of proteins which could be targeted in EGFR or other interconnected signaling cascades. To discover new targets we utilized two protein labeling techniques, stable isotope labeling by amino acids in cell culture (SILAC) and 10-plexed tandem mass tagging (TMT), combined with mass spectrometry quantification for measuring differences in protein expression in EGFR TKI resistant lung cancer cells compared to naïve parental cells. SILAC combined with mass spectrometry quantification, is an accurate method for quantifying differential changes in the cellular proteome. SILAC labeling involves the metabolic incorporation of “light” and “heavy” amino acids into proteins. In our study, to measure proteomic differences in resistant cells, H358 parental and resistant cells were grown in light and heavy SILAC media, respectively, containing 0.1mg/ml “light” 12C6L-lysine-2HCL and 12C6 14N4 L-Arginine-HCl or “heavy” 13C6 L-Lysine-2HCl and 13C6 15N4 L-Arginine-HCl with Dialyzed Fetal Bovine Serum. After six passages in SILAC media, cells were treated with/without TKIs erlotinib and with/without growth factor ligands EGF. Cell lysates were then collected, combined and run through an LC MS/MS Thermo LTQ-FTICR high resolution mass spectrometer. Differences in target proteins were identified and compared between heavy and light labeled peptides. Through this method, six proteins, including total-β-catenin, TPR, syntenin, HMGB2, TOM34 and AIF, were shown to be upregulated at least 1.5-fold in resistant cells and have the potential to play a role in EGFR TKI resistance. The TMT assay was also use in our study to analyze differential protein expression in both parental and resistant cells. The TMT protein expression analysis utilizes covalently bonded isobaric tags each of which produce a unique reporter ion signature which can be detected by mass spectrometry to differentiate between ten different samples. TMT was used to compare expression of key proteins in H358 cells in the presence or absence of TKIs erlotinib and growth factor ligands EGF. After sample preparation and lysate collection, proteins were reduced, alkylated, digested, and covalently labeled with isobaric tags for protein identification and quantitation by LC-MS analysis. In our study, GNA1 and SND1 were found via TMT in addition to proteins we found using SILAC to be upregulated and activated in resistant cells and their roles are currently being further investigated. In summary, this study compares two proteomic technologies and assesses their potential in determining novel cancer targets.

Author Details

Gregory Botting
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
Ichwaku Rastogi
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
Gagan Chhabra
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
Jason T Fong
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
Ryan J. Jacobs
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
Caleb Shearrow
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
David N. Moravec
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
Joseph Devito
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
Kymberly Harrington
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA
Ryan D. Bomgarden
Thermo Fisher Scientific, USA
John C. Rogers
Thermo Fisher Scientific, USA
Neelu Puri
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, USA

Citation

Gregory Botting, Ichwaku Rastogi, Gagan Chhabra, Jason T Fong, Ryan J. Jacobs, Caleb Shearrow, David N. Moravec, Joseph Devito, Kymberly Harrington, Ryan D. Bomgarden, John C. Rogers and Neelu Puri. Discovery of Biomarkers Mediating EGFR Tyrosine Kinase Inhibitor Resistance in Non-small Cell Lung Cancer: A Proteomics Approach. In Protein Purification: Principles and Trends. ISBN:978-1-922227-40-9. iConcept Press. 2016.

Download