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• W2170890534 abstract "Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ∼50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2–7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease. Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ∼50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2–7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease. Glioblastoma multiforme (GBM) 1The abbreviations used are:GBMGlioblastoma multiformeBICBayesian information criterionCAIIIcarbonic anhydrase IIIEGFREpidermal growth factor receptorEGFRvIIIEpidermal growth factor receptor variant IIIEMTEpithelial to mesenchymal transitionEps15Epidermal growth factor receptor substrate 15Gab1GRB2-associated-binding protein 1GBP1Guanylate-binding protein 1GOGene ontologyIEFIsoelectric fractionationIMACImmobilized metal affinity chromatographyIPImmunoprecipitationLC-MS/MSLiquid chromatography tandem mass spectrometryMVPMajor vault proteinNTRK1Neurotrophic receptor tyrosine kinasePANTHERProtein Analysis Through Evolutionary RelationshipsPDGFRPlatelet derived growth factor receptorPIP3Phosphatidylinositol_(3,4,5)-trisphosphatePLC-γPhospholipase C gammaPTENPhosphatase and tensin homologPTMPost-translational modificationREMBRANDTRepository for Molecular Brain Neoplasia DataRIPARadioimmunoprecipitation assayTCGAThe Cancer Genome AtlasWtWild type. 1The abbreviations used are:GBMGlioblastoma multiformeBICBayesian information criterionCAIIIcarbonic anhydrase IIIEGFREpidermal growth factor receptorEGFRvIIIEpidermal growth factor receptor variant IIIEMTEpithelial to mesenchymal transitionEps15Epidermal growth factor receptor substrate 15Gab1GRB2-associated-binding protein 1GBP1Guanylate-binding protein 1GOGene ontologyIEFIsoelectric fractionationIMACImmobilized metal affinity chromatographyIPImmunoprecipitationLC-MS/MSLiquid chromatography tandem mass spectrometryMVPMajor vault proteinNTRK1Neurotrophic receptor tyrosine kinasePANTHERProtein Analysis Through Evolutionary RelationshipsPDGFRPlatelet derived growth factor receptorPIP3Phosphatidylinositol_(3,4,5)-trisphosphatePLC-γPhospholipase C gammaPTENPhosphatase and tensin homologPTMPost-translational modificationREMBRANDTRepository for Molecular Brain Neoplasia DataRIPARadioimmunoprecipitation assayTCGAThe Cancer Genome AtlasWtWild type. is the most frequent and aggressive form of primary brain tumor (1Furnari F.B. Fenton T. Bachoo R.M. Mukasa A. Stommel J.M. Stegh A. Hahn W.C. Ligon K.L. Louis D.N. Brennan C. Chin L. DePinho R.A. Cavenee W.K. Malignant astrocytic glioma: genetics, biology, and paths to treatment.Genes Dev. 2007; 21: 2683-2710Crossref PubMed Scopus (1810) Google Scholar). The current standard of care for GBM consists of surgical removal, radiotherapy, and adjuvant chemotherapy (typically temozolomide) (1Furnari F.B. Fenton T. Bachoo R.M. Mukasa A. Stommel J.M. Stegh A. Hahn W.C. Ligon K.L. Louis D.N. Brennan C. Chin L. DePinho R.A. Cavenee W.K. Malignant astrocytic glioma: genetics, biology, and paths to treatment.Genes Dev. 2007; 21: 2683-2710Crossref PubMed Scopus (1810) Google Scholar). However, despite these interventions the prognosis is still poor, with mean survival time at ∼15 months following diagnosis (2Stupp R. Mason W.P. van den Bent M.J. Weller M. Fisher B. Taphoorn M.J.B. Belanger K. Brandes A.A. Marosi C. Bogdahn U. Curschmann J. Janzer R.C. Ludwin S.K. Gorlia T. Allgeier A. Lacombe D. Cairncross J.G. Eisenhauer E. Mirimanoff R.O. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.N. Engl. J. Med. 2005; 352: 987-996Crossref PubMed Scopus (14644) Google Scholar). Genetic profiling of GBM tumors has been used to identify multiple distinct genetic aberrations across a diverse array of genes such as the deletion of phosphatase and tensin homolog (PTEN), p16 deletion, and mutation of TP53 (3Kagawa N. Maruno M. Suzuki T. Hashiba T. Hashimoto N. Izumoto S. Yoshimine T. Detection of genetic and chromosomal aberrations in medulloblastomas and primitive neuroectodermal tumors with DNA microarrays.Brain Tumor Pathol. 2006; 23: 41-47Crossref PubMed Scopus (27) Google Scholar, 4Nobusawa S. Lachuer J. Wierinckx A. Kim Y.H. Huang J. Legras C. Kleihues P. Ohgaki H. Intratumoral patterns of genomic imbalance in glioblastomas.Brain Pathol. 2010; 20: 936-944PubMed Google Scholar). Additionally, amplification, overexpression, and/or mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) has been identified to be a key genetic alteration in ∼50% of GBM patients (5Network C.G.A.R. Comprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (5792) Google Scholar). EGFR amplification is often accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2–7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors (6Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity.Proc. Natl. Acad. Sci. 1994; 91: 7727-7731Crossref PubMed Scopus (813) Google Scholar, 7Ding H. Shannon P. Lau N. Wu X. Roncari L. Baldwin R.L. Takebayashi H. Nagy A. Gutmann D.H. Guha A. Oligodendrogliomas result from the expression of an activated mutant epidermal growth factor receptor in a RAS transgenic mouse astrocytoma model.Cancer Res. 2003; 63: 1106-1113PubMed Google Scholar, 8Wikstrand C.J. Hale L.P. Batra S.K. Hill M.L. Humphrey P.A. Kurpad S.N. McLendon R.E. Moscatello D. Pegram C.N. Reist C.J. Traweek S.T. Wong A.J. Zalutsky M.R. Bigner D.D. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas.Cancer Res. 1995; 55: 3140-3148PubMed Google Scholar). EGFRvIII is characterized by the deletion of exon 2–7, resulting in an in-frame deletion of 267 amino acid residues from the extracellular domain. This deletion generates a receptor which is unable to bind ligand yet is constitutively, but weakly, active (9Huang H.J.S. Nagane M. Klingbeil C.K. Lin H. Nishikawa R. Ji X.D. Huang C.M. Gill G.N. Wiley H.S. Cavenee W.K. The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers Is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling.J. Biol. Chem. 1997; 272: 2927-2935Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Continuous low level activation leads to impaired internalization and degradation of the receptor, causing prolonged signaling (10Schmidt M.H. Furnari F.B. Cavenee W.K. Bögler O. Epidermal growth factor receptor signaling intensity determines intracellular protein interactions, ubiquitination, and internalization.Proc. Natl. Acad. Sci. 2003; 100: 6505-6510Crossref PubMed Scopus (128) Google Scholar). Expression of EGFRvIII in the absence of wtEGFR leads to the transformation of cells in vivo, drives cell proliferation in vitro, and expression of EGFRvIII correlates with poor prognosis in the clinic (6Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity.Proc. Natl. Acad. Sci. 1994; 91: 7727-7731Crossref PubMed Scopus (813) Google Scholar, 11Batra S. Castelino-Prabhu S. Wikstrand C. Zhu X. Humphrey P. Friedman H. Bigner D. Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene.Cell Growth Differ. 1995; 6: 1251-1259PubMed Google Scholar, 12Shinojima N. Tada K. Shiraishi S. Kamiryo T. Kochi M. Nakamura H. Makino K. Saya H. Hirano H. Kuratsu J. Oka K. Ishimaru Y. Ushio Y. Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme.Cancer Res. 2003; 63: 6962-6970PubMed Google Scholar). EGFRvIII has been identified in GBM, lung, ovarian, and breast cancers, but has never been identified in normal tissue (13Garcia de Palazzo I.E. Adams G.P. Sundareshan P. Wong A.J. Testa J.R. Bigner D.D. Weiner L.M. Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas.Cancer Res. 1993; 53: 3217-3220PubMed Google Scholar, 14Moscatello D.K. Holgado-Madruga M. Godwin A.K. Ramirez G. Gunn G. Zoltick P.W. Biegel J.A. Hayes R.L. Wong A.J. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors.Cancer Res. 1995; 55: 5536-5539PubMed Google Scholar). Because of the absence of this mutant receptor in normal tissue, EGFRvIII is an attractive therapeutic target. Although EGFR inhibitors, such as erlotinib and gefitinib, inhibit EGFR, EGFRvIII bearing xenograft models and cell lines are resistant to these inhibitors (15Learn C.A. Hartzell T.L. Wikstrand C.J. Archer G.E. Rich J.N. Friedman A.H. Friedman H.S. Bigner D.D. Sampson J.H. Resistance to tyrosine kinase inhibition by mutant epidermal growth factor receptor variant III contributes to the neoplastic phenotype of glioblastoma multiforme.Clin. Cancer Res. 2004; 10: 3216-3224Crossref PubMed Scopus (143) Google Scholar, 16Pedersen M.W. Pedersen N. Ottesen L.H. Poulsen H.S. Differential response to gefitinib of cells expressing normal EGFR and the mutant EGFRvIII.Br. J. Cancer. 2005; 93: 915-923Crossref PubMed Scopus (59) Google Scholar). Therapeutic agents directly targeting EGFRvIII in murine GBM xenografts initially resulted in reduced tumor volume and a modest increase in survival (17Mishima K. Johns T.G. Luwor R.B. Scott A.M. Stockert E. Jungbluth A.A. Ji X.D. Suvarna P. Voland J.R. Old L.J. Huang H.J. Cavenee W.K. Growth suppression of intracranial xenografted glioblastomas overexpressing mutant epidermal growth factor receptors by systemic administration of monoclonal antibody (mAb) 806, a novel monoclonal antibody directed to the receptor.Cancer Res. 2001; 61: 5349-5354PubMed Google Scholar). However, tumor recurrence was inevitable because of resistance by uncharacterized evasion mechanisms and adaptations (17Mishima K. Johns T.G. Luwor R.B. Scott A.M. Stockert E. Jungbluth A.A. Ji X.D. Suvarna P. Voland J.R. Old L.J. Huang H.J. Cavenee W.K. Growth suppression of intracranial xenografted glioblastomas overexpressing mutant epidermal growth factor receptors by systemic administration of monoclonal antibody (mAb) 806, a novel monoclonal antibody directed to the receptor.Cancer Res. 2001; 61: 5349-5354PubMed Google Scholar). We propose that an improved understanding of the system-wide changes in protein expression and signaling caused by EGFRvIII expression should provide insight into specific therapeutic targets for EGFRvIII driven tumors. Glioblastoma multiforme Bayesian information criterion carbonic anhydrase III Epidermal growth factor receptor Epidermal growth factor receptor variant III Epithelial to mesenchymal transition Epidermal growth factor receptor substrate 15 GRB2-associated-binding protein 1 Guanylate-binding protein 1 Gene ontology Isoelectric fractionation Immobilized metal affinity chromatography Immunoprecipitation Liquid chromatography tandem mass spectrometry Major vault protein Neurotrophic receptor tyrosine kinase Protein Analysis Through Evolutionary Relationships Platelet derived growth factor receptor Phosphatidylinositol_(3,4,5)-trisphosphate Phospholipase C gamma Phosphatase and tensin homolog Post-translational modification Repository for Molecular Brain Neoplasia Data Radioimmunoprecipitation assay The Cancer Genome Atlas Wild type. Glioblastoma multiforme Bayesian information criterion carbonic anhydrase III Epidermal growth factor receptor Epidermal growth factor receptor variant III Epithelial to mesenchymal transition Epidermal growth factor receptor substrate 15 GRB2-associated-binding protein 1 Guanylate-binding protein 1 Gene ontology Isoelectric fractionation Immobilized metal affinity chromatography Immunoprecipitation Liquid chromatography tandem mass spectrometry Major vault protein Neurotrophic receptor tyrosine kinase Protein Analysis Through Evolutionary Relationships Platelet derived growth factor receptor Phosphatidylinositol_(3,4,5)-trisphosphate Phospholipase C gamma Phosphatase and tensin homolog Post-translational modification Repository for Molecular Brain Neoplasia Data Radioimmunoprecipitation assay The Cancer Genome Atlas Wild type. It is thought that EGFRvIIl enhances tumorigenicity by differential utilization (e.g. altered amplitude and kinetics and potentially novel components or pathways) of signal transduction pathways compared with ligand activated wtEGFR. Quantitative mass spectrometry has previously been applied to the identification of EGFRvIII specific phosphotyrosine signaling across four GBM cell lines expressing titrated levels of EGFRvIII relative to cells expressing the kinase-dead control (18Huang P.H. Mukasa A. Bonavia R. Flynn R.A. Brewer Z.E. Cavenee W.K. Furnari F.B. White F.M. Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma.Proc. Natl. Acad. Sci. 2007; 104: 12867-12872Crossref PubMed Scopus (320) Google Scholar). Cross-activation of EGFRvIII and the c-Met receptor tyrosine kinase is prevalent within these EGFRvIII overexpressing cell lines, revealing an attractive therapeutic strategy (18Huang P.H. Mukasa A. Bonavia R. Flynn R.A. Brewer Z.E. Cavenee W.K. Furnari F.B. White F.M. Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma.Proc. Natl. Acad. Sci. 2007; 104: 12867-12872Crossref PubMed Scopus (320) Google Scholar), which was later extended to include cross-activation of PDGFR (platelet-derived growth factor receptor) (19Stommel J.M. Kimmelman A.C. Ying H. Nabioullin R. Ponugoti A.H. Wiedemeyer R. Stegh A.H. Bradner J.E. Ligon K.L. Brennan C. Chin L. DePinho R.A. Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies.Science. 2007; 318: 287-290Crossref PubMed Scopus (753) Google Scholar). Although EGFRvIII signaling has been extensively studied in GBM cell lines, the molecular mechanisms of increased tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated (20Huang P.H. Miraldi E.R. Xu A.M. Kundukulam V.A. Del Rosario A.M. Flynn R.A. Cavenee W.K. Furnari F.B. White F.M. Phosphotyrosine signaling analysis of site-specific mutations on EGFRvIII identifies determinants governing glioblastoma cell growth.Mol. BioSystems. 2010; 6: 1227-1237Crossref PubMed Scopus (39) Google Scholar, 21Chumbalkar V. Latha K. Hwang Y. Maywald R. Hawley L. Sawaya R. Diao L. Baggerly K. Cavenee W.K. Furnari F.B. Bogler O. Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of ΔEGFR.J. Proteome Res. 2011; 10: 1343-1352Crossref PubMed Scopus (38) Google Scholar). In addition, tissue culture conditions dramatically change the genetic and molecular characteristics found in primary human tumors. In particular, EGFRvIII expression is rapidly lost during generation of primary culture cells from GBM tumors. Most of the EGFRvIII-expressing cells lines are a result of stable transfection, rather than endogenous expression, of the mutant receptor (22Pandita A. Aldape K.D. Zadeh G. Guha A. James C.D. Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR.Gene. Chromosomes Cancer. 2004; 39: 29-36Crossref PubMed Scopus (188) Google Scholar). Additionally, the micro-environment and cellular heterogeneity of the tumor have a significant impact on the response to therapeutics, yet are poorly reflected in cell culture. As a consequence, quantification of signaling networks in glioblastoma cell lines provide a limited understanding of the signaling networks in GBM tumor samples. To overcome this limitation, the James and Sarkaria labs have generated, from patient surgical specimens, a panel of glioblastoma tumor xenografts that are maintained through serial passaging as subcutaneous xenografts in nude mice (22Pandita A. Aldape K.D. Zadeh G. Guha A. James C.D. Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR.Gene. Chromosomes Cancer. 2004; 39: 29-36Crossref PubMed Scopus (188) Google Scholar, 23Carlson B.L. Pokorny J.L. Schroeder M.A. Sarkaria J.N. Establishment, maintenance, and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.Curr. Protoc. Pharmacol. 2011; 52: 14.16.23-14.16.51Crossref Scopus (125) Google Scholar). Maintenance of GBM tumors in this in vivo setting preserves the genetic features and phenotypes crucial to the tumorigenicity of the primary human tumors (23Carlson B.L. Pokorny J.L. Schroeder M.A. Sarkaria J.N. Establishment, maintenance, and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.Curr. Protoc. Pharmacol. 2011; 52: 14.16.23-14.16.51Crossref Scopus (125) Google Scholar). With these tumor xenografts it is possible to analyze in vivo signaling networks, predict optimal therapeutic strategies based on these data, and test these predictions in a physiologically relevant system. To quantify signaling networks activated in glioblastoma tumor xenografts and determine the effect of wtEGFR or EGFRvIII expression on these networks, we applied quantitative mass spectrometry to eight human GBM xenografts expressing wtEGFR (wt) or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+) implanted into the flanks of nude mice. This analysis led to the identification and quantification of 1588 proteins (across two or more biological replicates) and 225 tyrosine phosphorylation sites on 168 proteins across eight tumor xenografts. Integration of quantitative phosphotyrosine data and protein expression profiles have uncovered the differential regulation of novel proteins and phosphotyrosine sites, which relate to the mode of action of wtEGFR and EGFRvIII overexpression in vivo. Quantification of tyrosine phosphorylation networks revealed signaling specific to each tumor xenograft. These data provide evidence for a significant amount of variation across the eight xenografts, and suggests that optimal therapeutic strategies might be specific to each tumor. Human GBM xenografts were established with the ectopic injection of 100–200 μl of tumor homogenate mixed 1:1 with matrigel into the flanks of nude mice (Fig. 1A). Each of the eight xenografts used in this study (GBM6, GBM8, GBM10, GBM12, GBM15, GBM26, GBM39, and GBM59) were derived from primary tumors from different patients undergoing surgical treatment and serially passaged in mice before this study (23Carlson B.L. Pokorny J.L. Schroeder M.A. Sarkaria J.N. Establishment, maintenance, and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.Curr. Protoc. Pharmacol. 2011; 52: 14.16.23-14.16.51Crossref Scopus (125) Google Scholar). Four tumor xenografts were generated for each GBM tumor except for GBM6 where two tumor xenografts were generated. Tumor xenografts were resected from mice and immediately flash frozen and stored in liquid nitrogen prior to tissue homogenization. Tumor xenograft tissues were homogenized (Polytron) in ice-cold 8 m urea, 1 mm sodium orthovanadate, 0.1% Nonidet P-40, and protease and phosphatase inhibitor mixture tablets (Roche) for mass spectrometric analyses. For immunoblotting, ice-cold radioimmunoprecipitation assay (RIPA) buffer plus 1 mm sodium orthovanadate, 0.1% Nonidet P-40, and protease and phosphatase inhibitor mixture tablets (Roche) was used for homogenization. Samples were homogenized on ice, with 6 × 10 s pulses (full speed), separated by 10 s intervals to prevent heating of the sample. Once homogenization was complete tumor lysates were left to settle on ice for 2 min. A 50 μl aliquot was taken for BCA protein quantification and the total homogenate was stored at −80 °C. Homogenized tumor lysates (generated as described above) were separated on a 7.5% polyacrylamide gel and electrophoretically transferred to nitrocellulose (Bio-Rad). Blocking buffers were made in TBS-T (150 mm NaCl, 0.1% Tween 20, 50 mm Tris, pH 8.0) and contained 5% nonfat dry milk or 3% bovine serum albumin. Antibodies used are as follows; Anti-EGFR (BD Biosciences), Anti-GAPDH (CST, Danvers, MA), Anti-phosphotyrosine (4G10, Millipore), Anti-Cbl (CST), Anti-GEFH1 (CST), Anti-Hrs (Enzo Life Sciences, Farmingdale, NY), Anti-Shp2 (CST), Anti-EGFR pY1173 (CST), Anti-EGFR pY1045 (CST), Anti-STAT3 (CST), Anti-STAT3 pY705 (CST), Anti-PLC-γ (BD Biosciences), Anti-Gab1 (Millipore), and Anti-Gab1 pY627 (Millipore). Appropriate antibodies were diluted in blocking buffer and incubated with nitrocellulose overnight at 4 °C. Secondary antibodies (either goat anti-rabbit or goat anti-mouse conjugated to horseradish peroxidise) were diluted 1/10,000 in TBS-T and incubated at room temperature for 1 h. Antibody binding was detected using the enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL). Proteins were quantified using BCA assay (Pierce) and proteins were reduced (10 mm dithiothreitol, 56 °C for 45min), alkylated (50 mm iodoacetamide, room temperature in the dark for 1 h), and excess iodoaceamide was quenched with dithiothreitol to a final concentration of 25 mm. Protein was subsequently digested with trypsin (sequencing grade, Promega, Madison, WI), at an enzyme/substrate ratio of 1:100, at room temperature overnight in 100 mm ammonium acetate pH 8.9. Trypsin activity was quenched by adding formic acid to a final concentration of 5%. Urea was removed from the samples by reverse phase desalting using a C18 cartridge (Waters, Milford, MA) and peptides were lyophilized and stored at −80 °C. Peptide labeling with iTRAQ 8plex (AB Sciex) was performed as previously described (24Zhang Y. Wolf-Yadlin A. Ross P.L. Pappin D.J. Rush J. Lauffenburger D.A. White F.M. Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules.Mol. Cell. Proteomics. 2005; 4: 1240-1250Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). Briefly, for each analysis, ∼8 mg (wet weight) tumor (equivalent to 800 μg peptide before desalting and processing) for each of the eight tumors was labeled with two tubes of iTRAQ 8plex reagent. GBM xenografts were labeled using the iTRAQ 8plex channels as follows: 113-GBM6; 114-GBM8; 115-GBM10; 116-GBM12; 117-GBM15; 118-GBM26; 119-GBM39; and 121-GBM59 throughout all four biological replicates. GBM6 biological replicate 1 and biological replicate 4 were analyzed in the place of biological replicate 2 and 3 respectively. Lyophilized samples were dissolved in 60 μl of 500 mm triethylammonium bicarbonate, pH 8.5, and the iTRAQ reagent was dissolved in 70 μl of isopropanol. The solution containing peptides and iTRAQ reagent was vortexed, incubated at room temperature for 2 h and concentrated to 40 μl. Samples labeled with eight different isotopic iTRAQ reagents were combined and concentrated to completion. Peptides were then dissolved in 400 μl of IP buffer (100 mm Tris, 100 mm NaCl, and 1% Nonidet P-40, pH 7.4) and the pH was adjusted to 7.4 before phosphotyrosine immunoprecipitation (IP). Protein G agarose (80 μl, EMD) was incubated with three phosphotyrosine antibodies; 12 μg PT66 (Sigma-Aldrich), 12 μg pY100 (CST), and 12 μg 4G10 (Millipore) and 200 μl of IP buffer (100 mm Tris, 1% Nonidet P-40, pH 7.4) was added and the mixture was incubated for 8 h at 4 °C with rotation. Antibody conjugated protein G was then rinsed and iTRAQ 8plex labeled peptides re-suspended in the IP buffer, added to the conjugated protein G and incubated overnight at 4 °C with rotation. Conjugated protein G agarose was rinsed with 400 μl of IP buffer and 4 × 400 μl of rinse buffer (100 mm Tris, pH 7.4), and peptides were eluted into 70 μl of 100 mm glycine pH 2. Phosphotyrosine peptides were further enriched using an offline immobilized metal affinity chromatography (IMAC) column (24Zhang Y. Wolf-Yadlin A. Ross P.L. Pappin D.J. Rush J. Lauffenburger D.A. White F.M. Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules.Mol. Cell. Proteomics. 2005; 4: 1240-1250Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). Peptides were loaded onto a pre-column and were subsequently separated by reverse phase HPLC (Agilent, Santa Clara, CA) over a 150 min gradient before nanoelectrospray into an Orbitrap XL mass spectrometer (Thermo scientific) for phosphotyrosine analyses. To correct for slight variations in the amount of sample in each of the iTRAQ channels, the mean iTRAQ ratios for all proteins identified in each biological replicate analysis was used to normalize the data. The mass spectrometer was operated in data-dependent mode with a full scan MS spectrum followed by MS/MS (collision-induced dissociation (CID) was set at 35% energy for sequence information and higher-energy c-trap dissociation (HCD) at 75% energy for iTRAQ quantification for Orbitrap XL) for the top 10 precursor ions in each cycle. Ion trap injection time was set to 100 ms and FTMS injection time was set to 1000 ms with a resolution of 60,000 across m/z 400–2000. For IT and FT-MS/MS scans, fragmentation was carried out on ions above a threshold of 500 counts and an FTMS resolution of 7500. Raw mass spectral data files (.RAW files) were converted into .mgf file format using DTAsupercharge 1.31 (http://msquant.sourceforge.net/). All resulting MS/MS peak lists were searched against a NCBI UniProt 2009 database containing Homo sapiens protein sequences (37,743 entries) using Mascot version: 2.1.03 (Matrix Science). Briefly, trypsin enzyme specificity was applied with a maximum of 1 missed cleavage. Mass tolerance for precursor ions was set to 10 ppm and fragment ion mass tolerance was 0.8 Da. MS/MS spectra searches incorporated fixed modifications of carbamidomethylation of cysteine and iTRAQ 8plex modification of lysines and peptide N termini. Variable modifications were oxidized methionine, and phosphorylation of serine, threonine, and tyrosine residues. Phosphoty" @default.
• W2170890534 created "2016-06-24" @default.
• W2170890534 creator A5000847750 @default.
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• W2170890534 date "2012-12-01" @default.
• W2170890534 modified "2023-10-16" @default.
• W2170890534 title "Molecular Characterization of EGFR and EGFRvIII Signaling Networks in Human Glioblastoma Tumor Xenografts" @default.
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