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Phosphotyrosine Profiling of NSCLC Cells in Response to EGF and HGF Reveals Network Specific Mediators of Invasion

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† Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
‡ Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
§ Tailored Therapeutics, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, United States
*Phone: (617) 258-8949. Fax: (617) 452-4978. E-mail: [email protected]
Cite this: J. Proteome Res. 2013, 12, 4, 1856–1867
Publication Date (Web):February 26, 2013
https://doi.org/10.1021/pr301192t
Copyright © 2013 American Chemical Society
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    Abstract

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    Growth factor signaling is deregulated in cancer and often leads to invasion, yet receptor tyrosine kinase signaling pathways driving invasion under different growth factor conditions are not well understood. To identify specific signaling molecules regulating invasion of A549 non-small cell lung carcinoma (NSCLC) cells downstream of the epidermal growth factor receptor (EGFR) and Met, quantitative site-specific mass spectrometric analysis of tyrosine phosphorylation was performed following epidermal growth factor (EGF) or hepatocyte growth factor (HGF) stimulation, at three different growth factor concentrations and at two time points. Through this analysis, the temporal and concentration dependent phosphorylation profiles were obtained for 131 and 139 sites downstream of EGF and HGF stimulation, respectively. To characterize the effect of these signaling network alterations, we quantified 3D cell migration/invasion through Matrigel. Partial least-squares regression (PLSR) analysis was performed to identify the tyrosine phosphorylation sites most strongly correlated with EGF and/or HGF mediated invasion. Potential common and specific signaling events required for driving invasion downstream of EGFR and Met were identified using either a combined or two independent PLSR models, based on the quantitative EGF or HGF data. Our data highlight the integration and compartmentalization of signaling required for invasion in cancer.

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    Supplementary Figure 1: Manually validated phosphotyrosine peptides with confirmed site localization for the EGF data set comprised of 3 biological replicates. The x-axis represents the m/z ratio and the y-axis represents the intensity of the fragment ions. The precursor ion selection window is displayed at the top right-hand side of each page for each indicated peptide sequence and the isolation window (± 1 m/z) is shaded in gray. Peptides were discarded if there were any ions in a window of ±1.5 m/z with intensity above 25% of the intended precursor ion. The iTRAQ reporter ion window is shown for each indicated peptide at the bottom right-hand corner of each MS/MS spectrum. y and b-series ions are indicated in red on the peptide sequence at the top of each page and labeled at the top of each fragment ion. Fragment ions highlighted in green are 0.01% away from the predicted m/z and those highlighted in magenta are 0.015% away from the predicted m/z. Supplementary Figure 2: Manually validated phosphotyrosine peptides with confirmed site localization for the HGF data set comprised of 3 biological replicates. The x-axis represents the m/z ratio and the y-axis represents the intensity of the fragment ions. The precursor ion selection window is displayed at the top right-hand side of each page for each indicated peptide sequence and the isolation window (± 1 m/z) is shaded in gray. Peptides were discarded if there were any ions in a window of ±1.5 m/z with an intensity above 25% of the intended precursor ion. The iTRAQ reporter ion window is shown for each indicated peptide at the bottom right-hand corner of each MS/MS spectrum. y and b-series ions are indicated in red on the peptide sequence at the top of each page and labeled at the top of each fragment ion. Fragment ions highlighted in green are 0.01% away from the predicted m/z and those highlighted in magenta are 0.015% away from the predicted m/z. Supplementary Table 1: Summary of 131 peptides identified and quantified across A549 cells stimulated with 3 different concentrations of EGF at 2 different time points. The biological replicates in which each of the identified peptides were quantified are indicated as a, b and/or c. The abbreviated name and phosphorylation site location with respect to the full protein sequence is shown. The phosphorylation site(s) are indicated in the peptide sequence as a lower case y (tyrosine) and s (serine) or t (threonine) (for multiply phosphorylated peptides). The observed m/z is shown with a representative Mascot score. Quantitative iTRAQ ratios are shown normalized to 10% FBS with the standard deviation and coefficients of variation (where applicable). The affinity propagation cluster is indicated for each peptide followed by the iTRAQ ratios normalized to the mean of all 8 channels and then normalized to the mean of all 8 channels and Log2 transformed. Supplementary Table 2: Summary of 139 peptides identified and quantified across A549 cells stimulated with 3 different concentrations of HGF at 2 different time points. The biological replicates in which each of the identified peptides were quantified are indicated as a, b and/or c. The abbreviated name and phosphorylation site location with respect to the full protein sequence is shown. The phosphorylation site(s) are indicated in the peptide sequence as a lower case y (tyrosine) and s (serine) or t (threonine) (for multiply phosphorylated peptides). The observed m/z is shown with a representative Mascot score. Quantitative iTRAQ ratios are shown normalized to 10% FBS with the standard deviation and coefficients of variation (where applicable). The affinity propagation cluster is indicated for each peptide followed by the iTRAQ ratios normalized to the mean of all 8 channels and then normalized to the mean of all 8 channels and Log2 transformed. This material is available free of charge via the Internet at http://pubs.acs.org.

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