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Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Autelitano F, Loyaux D, Roudières S, Déon C, Guette F, Fabre P, Ping Q, Wang S, Auvergne R, Badarinarayana V, Smith M, Guillemot JC, Goldman SA, Natesan S, Ferrara P, August P - PLoS ONE (2014)

Bottom Line: Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need".Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Sanofi-Aventis Recherche & Développement, Centre de Toulouse, Toulouse, France.

ABSTRACT
Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

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Profiling cell surface sialoglycoproteins via the bio-orthogonal chemical reporter strategy combined with quantitative shotgun proteomics.The strategy consists of six steps: 1) metabolic labeling of cells with Ac4ManNAz; 2) chemoselective conjugation of azide-labeled glycans with a biotin-linked phosphine; 3) cell lysis; 4) affinity enrichment of the biotin-tagged proteins on streptavidin-conjugated beads; 5) on-beads trypsin digestion of affinity-captured sialoglycoproteins; 6) identification using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a nano-LC-LTQ Orbitrap mass spectrometer platform and sequence database searching, and quantitative differential analysis of the sialoglycoproteins using label-free analysis with DIFFTAL (DIFferential Fourier-Transform AnaLysis) software algorithm.
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pone-0110316-g001: Profiling cell surface sialoglycoproteins via the bio-orthogonal chemical reporter strategy combined with quantitative shotgun proteomics.The strategy consists of six steps: 1) metabolic labeling of cells with Ac4ManNAz; 2) chemoselective conjugation of azide-labeled glycans with a biotin-linked phosphine; 3) cell lysis; 4) affinity enrichment of the biotin-tagged proteins on streptavidin-conjugated beads; 5) on-beads trypsin digestion of affinity-captured sialoglycoproteins; 6) identification using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a nano-LC-LTQ Orbitrap mass spectrometer platform and sequence database searching, and quantitative differential analysis of the sialoglycoproteins using label-free analysis with DIFFTAL (DIFferential Fourier-Transform AnaLysis) software algorithm.

Mentions: In this work, proteomic profiling was performed employing BOCR technology in combination with LFQ-MS to accurately identify and quantify the individual cell surface sialoglycoproteome in human glioma tissues. We applied the strategy schematized in Fig. 1 to analyze differentially expressed cell surface sialoglycoproteins in primary cultures derived from GBM patient tumors, fetal and adult human astrocytes, as well as human NPCs. Several technical and scientific questions were addressed in this study. From the technical prospective, experiments were performed to determine if the BOCR strategy in combination with LFQ-MS provides accuracy and sensitivity enough to allow robust identification of the surface antigens or receptors already validated for the targeted therapy of HGG. More importantly, we addressed whether this technological advancement could facilitate the discovery of new tumor-associated cell surface antigens or receptors to enable better specificity for the targeting of malignant gliomas. Importantly, the effectiveness of our technical and scientific approach was validated by the identification of well-known glioma therapeutic targets and new cell surface antigens whose expression is predominantly restricted to GBM tumors which was confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. Altogether, this result demonstrates the translational power of our quantitative sialoglyproteomics approach for the discovery of new credentialed biomarkers and therapeutic targets for treatment of malignant gliomas.


Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Autelitano F, Loyaux D, Roudières S, Déon C, Guette F, Fabre P, Ping Q, Wang S, Auvergne R, Badarinarayana V, Smith M, Guillemot JC, Goldman SA, Natesan S, Ferrara P, August P - PLoS ONE (2014)

Profiling cell surface sialoglycoproteins via the bio-orthogonal chemical reporter strategy combined with quantitative shotgun proteomics.The strategy consists of six steps: 1) metabolic labeling of cells with Ac4ManNAz; 2) chemoselective conjugation of azide-labeled glycans with a biotin-linked phosphine; 3) cell lysis; 4) affinity enrichment of the biotin-tagged proteins on streptavidin-conjugated beads; 5) on-beads trypsin digestion of affinity-captured sialoglycoproteins; 6) identification using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a nano-LC-LTQ Orbitrap mass spectrometer platform and sequence database searching, and quantitative differential analysis of the sialoglycoproteins using label-free analysis with DIFFTAL (DIFferential Fourier-Transform AnaLysis) software algorithm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4216004&req=5

pone-0110316-g001: Profiling cell surface sialoglycoproteins via the bio-orthogonal chemical reporter strategy combined with quantitative shotgun proteomics.The strategy consists of six steps: 1) metabolic labeling of cells with Ac4ManNAz; 2) chemoselective conjugation of azide-labeled glycans with a biotin-linked phosphine; 3) cell lysis; 4) affinity enrichment of the biotin-tagged proteins on streptavidin-conjugated beads; 5) on-beads trypsin digestion of affinity-captured sialoglycoproteins; 6) identification using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a nano-LC-LTQ Orbitrap mass spectrometer platform and sequence database searching, and quantitative differential analysis of the sialoglycoproteins using label-free analysis with DIFFTAL (DIFferential Fourier-Transform AnaLysis) software algorithm.
Mentions: In this work, proteomic profiling was performed employing BOCR technology in combination with LFQ-MS to accurately identify and quantify the individual cell surface sialoglycoproteome in human glioma tissues. We applied the strategy schematized in Fig. 1 to analyze differentially expressed cell surface sialoglycoproteins in primary cultures derived from GBM patient tumors, fetal and adult human astrocytes, as well as human NPCs. Several technical and scientific questions were addressed in this study. From the technical prospective, experiments were performed to determine if the BOCR strategy in combination with LFQ-MS provides accuracy and sensitivity enough to allow robust identification of the surface antigens or receptors already validated for the targeted therapy of HGG. More importantly, we addressed whether this technological advancement could facilitate the discovery of new tumor-associated cell surface antigens or receptors to enable better specificity for the targeting of malignant gliomas. Importantly, the effectiveness of our technical and scientific approach was validated by the identification of well-known glioma therapeutic targets and new cell surface antigens whose expression is predominantly restricted to GBM tumors which was confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. Altogether, this result demonstrates the translational power of our quantitative sialoglyproteomics approach for the discovery of new credentialed biomarkers and therapeutic targets for treatment of malignant gliomas.

Bottom Line: Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need".Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Sanofi-Aventis Recherche & Développement, Centre de Toulouse, Toulouse, France.

ABSTRACT
Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Show MeSH
Related in: MedlinePlus