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Enhanced Validation of Antibodies Enables the Discovery of Missing Proteins.

ABSTRACT: The localization of proteins at a tissue- or cell-type-specific level is tightly linked to the protein function. To better understand each protein's role in cellular systems, spatial information constitutes an important complement to quantitative data. The standard methods for determining the spatial distribution of proteins in single cells of complex tissue samples make use of antibodies. For a stringent analysis of the human proteome, we used orthogonal methods and independent antibodies to validate 5981 antibodies that show the expression of 3775 human proteins across all major human tissues. This enhanced validation uncovered 56 proteins corresponding to the group of "missing proteins" and 171 proteins of unknown function. The presented strategy will facilitate further discussions around criteria for evidence of protein existence based on immunohistochemistry and serves as a useful guide to identify candidate proteins for integrative studies with quantitative proteomics methods.

SUBMITTER: Sivertsson A

PROVIDER: S-EPMC7723238 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Enhanced Validation of Antibodies Enables the Discovery of Missing Proteins.

Sivertsson Åsa Å Lindström Emil E Oksvold Per P Katona Borbala B Hikmet Feria F Vuu Jimmy J Gustavsson Jonas J Sjöstedt Evelina E von Feilitzen Kalle K Kampf Caroline C Schwenk Jochen M JM Uhlén Mathias M Lindskog Cecilia C

Journal of proteome research 20201110 12

The localization of proteins at a tissue- or cell-type-specific level is tightly linked to the protein function. To better understand each protein's role in cellular systems, spatial information constitutes an important complement to quantitative data. The standard methods for determining the spatial distribution of proteins in single cells of complex tissue samples make use of antibodies. For a stringent analysis of the human proteome, we used orthogonal methods and independent antibodies to va ...[more]

PMID: 33170010

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Project description:BackgroundWnt signaling is an evolutionarily conserved developmental pathway that is frequently hyperactivated in cancer. While multiple protein-coding genes regulated by Wnt signaling are known, the functional lncRNAs regulated by Wnt signaling have not been systematically characterized.MethodsWe comprehensively mapped Wnt-regulated lncRNAs from an orthotopic Wnt-addicted pancreatic cancer model and examined the response of lncRNAs to Wnt inhibition between in vivo and in vitro cancer models. We further annotated and characterized these Wnt-regulated lncRNAs using existing genomic classifications (using data from FANTOM5) in the context of Wnt signaling and inferred their role in cancer pathogenesis (using GWAS and expression data from the TCGA). To functionally validate Wnt-regulated lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation both in vivo and in vitro.ResultsWe identified 3633 lncRNAs, of which 1503 were regulated by Wnt signaling in an orthotopic Wnt-addicted pancreatic cancer model. These lncRNAs were much more sensitive to changes in Wnt signaling in xenografts than in cultured cells. Our analysis suggested that Wnt signaling inhibition could influence the co-expression relationship of Wnt-regulated lncRNAs and their eQTL-linked protein-coding genes. Wnt-regulated lncRNAs were also implicated in specific gene networks involved in distinct biological processes that contribute to the pathogenesis of cancers. Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1503) of the Wnt-regulated lncRNAs were found to modify cancer cell growth in vitro. This included CCAT1 and LINC00263, previously reported to regulate cancer growth. Using an in vivo CRISPRi screen, we doubled the discovery rate, identifying twice as many Wnt-regulated lncRNAs (25/1503) that had a functional effect on cancer cell growth.ConclusionsOur study demonstrates the value of studying lncRNA functions in vivo, provides a valuable resource of lncRNAs regulated by Wnt signaling, and establishes a framework for systematic discovery of functional lncRNAs.

Dataset Information

Enhanced Validation of Antibodies Enables the Discovery of Missing Proteins.

Publications

Enhanced Validation of Antibodies Enables the Discovery of Missing Proteins.

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