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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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Classification of identified sialoglycoproteins by GO annotation.(A) Cellular components were assigned using Ingenuity Pathway Analysis Knowledge Base (http://ingenuity.com/), the Human Protein Reference Database (http://www.hprd.org/), and the Gene Ontology (GO) Consortium (http://geneontology.org/). (B) Molecular functions were assigned using the PANTHER classification system (http://www.pantherdb.org/). Of the 843 unique sialoglycoproteins confidently identified from the ten samples, 485 (58%) and 121 (14%) proteins could be assigned to the “plasma membrane” (GO:0005886) and “extracellular space” (GO:0005615) GO categories, respectively. A total of 819 molecular function hits were allotted to the 606 cell surface proteins. Of these, approximately one-third (34%) correspond to receptor activity including all major classes of cell surface receptor proteins.
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pone-0110316-g005: Classification of identified sialoglycoproteins by GO annotation.(A) Cellular components were assigned using Ingenuity Pathway Analysis Knowledge Base (http://ingenuity.com/), the Human Protein Reference Database (http://www.hprd.org/), and the Gene Ontology (GO) Consortium (http://geneontology.org/). (B) Molecular functions were assigned using the PANTHER classification system (http://www.pantherdb.org/). Of the 843 unique sialoglycoproteins confidently identified from the ten samples, 485 (58%) and 121 (14%) proteins could be assigned to the “plasma membrane” (GO:0005886) and “extracellular space” (GO:0005615) GO categories, respectively. A total of 819 molecular function hits were allotted to the 606 cell surface proteins. Of these, approximately one-third (34%) correspond to receptor activity including all major classes of cell surface receptor proteins.

Mentions: A total of 843 unique proteins including 786 N-linked glycoproteins were confidently identified and quantified from the seven samples by DIFFTAL (DIFferential Fourier-Transform AnaLysis) software algorithm [37] and gene ontology (GO)-annotated using the Ingenuity Pathway Analysis Knowledge Base (http://ingenuity.com/), the Human Protein Reference Database (http://www.hprd.org/) [38], the Gene Ontology Consortium (http://geneontology.org/) and the UniProtKB/Swiss-Prot Protein Knowledgebase (http://www.uniprot.org/). The number of N-linked glycoproteins identified is certainly underestimated since the use of the UniProtKB/Swiss-Prot database to determine if a protein is glycosylated is notoriously under-representative. Table S1 lists the information of the 843 proteins identified from GBM tumor cells, fetal and adult astrocytes and NPCs. Of these proteins, 485 (58%) and 121 (14%) could be assigned to the “plasma membrane” and “extracellular space” GO categories, respectively, whereas 237 (28%) proteins were annotated as “intracellular” including 205 (24%) “integral to membrane” (GO:0016021) or “membrane-bounded organelle” proteins, 12 (∼1%) “lysosomal” proteins (GO:0005764) and 16 (∼2%) proteins assigned to “organelle lumens” or “cytoplasm” (Fig. 5A and Table S1). Interestingly, 601 (87%) of the 690 membrane-associated proteins and 16 (∼13%) of the 121 proteins annotated “extracellular space” contain at least one predicted TM segment and therefore are likely to be constituents of the plasma membrane. Finally, 708 (84%) of the 843 sialoglycoproteins identified in our study were predicted by the SignalP 3.0 software (http://cbs.dtu.ck/services/) to contain a signal peptide. These data suggest there was a significant enrichment of the pool of cell surface sialoglycosylated proteins using this method.


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)

Classification of identified sialoglycoproteins by GO annotation.(A) Cellular components were assigned using Ingenuity Pathway Analysis Knowledge Base (http://ingenuity.com/), the Human Protein Reference Database (http://www.hprd.org/), and the Gene Ontology (GO) Consortium (http://geneontology.org/). (B) Molecular functions were assigned using the PANTHER classification system (http://www.pantherdb.org/). Of the 843 unique sialoglycoproteins confidently identified from the ten samples, 485 (58%) and 121 (14%) proteins could be assigned to the “plasma membrane” (GO:0005886) and “extracellular space” (GO:0005615) GO categories, respectively. A total of 819 molecular function hits were allotted to the 606 cell surface proteins. Of these, approximately one-third (34%) correspond to receptor activity including all major classes of cell surface receptor proteins.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110316-g005: Classification of identified sialoglycoproteins by GO annotation.(A) Cellular components were assigned using Ingenuity Pathway Analysis Knowledge Base (http://ingenuity.com/), the Human Protein Reference Database (http://www.hprd.org/), and the Gene Ontology (GO) Consortium (http://geneontology.org/). (B) Molecular functions were assigned using the PANTHER classification system (http://www.pantherdb.org/). Of the 843 unique sialoglycoproteins confidently identified from the ten samples, 485 (58%) and 121 (14%) proteins could be assigned to the “plasma membrane” (GO:0005886) and “extracellular space” (GO:0005615) GO categories, respectively. A total of 819 molecular function hits were allotted to the 606 cell surface proteins. Of these, approximately one-third (34%) correspond to receptor activity including all major classes of cell surface receptor proteins.
Mentions: A total of 843 unique proteins including 786 N-linked glycoproteins were confidently identified and quantified from the seven samples by DIFFTAL (DIFferential Fourier-Transform AnaLysis) software algorithm [37] and gene ontology (GO)-annotated using the Ingenuity Pathway Analysis Knowledge Base (http://ingenuity.com/), the Human Protein Reference Database (http://www.hprd.org/) [38], the Gene Ontology Consortium (http://geneontology.org/) and the UniProtKB/Swiss-Prot Protein Knowledgebase (http://www.uniprot.org/). The number of N-linked glycoproteins identified is certainly underestimated since the use of the UniProtKB/Swiss-Prot database to determine if a protein is glycosylated is notoriously under-representative. Table S1 lists the information of the 843 proteins identified from GBM tumor cells, fetal and adult astrocytes and NPCs. Of these proteins, 485 (58%) and 121 (14%) could be assigned to the “plasma membrane” and “extracellular space” GO categories, respectively, whereas 237 (28%) proteins were annotated as “intracellular” including 205 (24%) “integral to membrane” (GO:0016021) or “membrane-bounded organelle” proteins, 12 (∼1%) “lysosomal” proteins (GO:0005764) and 16 (∼2%) proteins assigned to “organelle lumens” or “cytoplasm” (Fig. 5A and Table S1). Interestingly, 601 (87%) of the 690 membrane-associated proteins and 16 (∼13%) of the 121 proteins annotated “extracellular space” contain at least one predicted TM segment and therefore are likely to be constituents of the plasma membrane. Finally, 708 (84%) of the 843 sialoglycoproteins identified in our study were predicted by the SignalP 3.0 software (http://cbs.dtu.ck/services/) to contain a signal peptide. These data suggest there was a significant enrichment of the pool of cell surface sialoglycosylated proteins using this method.

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