| 1. |
- Sjöstedt, Evelina, et al.
(författare)
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Integration of Transcriptomics and Antibody-Based Proteomics for Exploration of Proteins Expressed in Specialized Tissues
- 2018
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 17:12, s. 4127-4137
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Tidskriftsartikel (refereegranskat)abstract
- A large portion of human proteins are referred to as missing proteins, defined as protein-coding genes that lack experimental data on the protein level due to factors such as temporal expression, expression in tissues that are difficult to sample, or they actually do not encode functional proteins. In the present investigation, an integrated omics approach was used for identification and exploration of missing proteins. Transcriptomics data from three different sourcesthe Human Protein Atlas (HPA), the GTEx consortium, and the FANTOM5 consortiumwere used as a starting point to identify genes selectively expressed in specialized tissues. Complementing the analysis with profiling on more specific tissues based on immunohistochemistry allowed for further exploration of cell-type-specific expression patterns. More detailed tissue profiling was performed for >300 genes on complementing tissues. The analysis identified tissue-specific expression of nine proteins previously listed as missing proteins (POU4F1, FRMD1, ARHGEF33, GABRG1, KRTAP2-1, BHLHE22, SPRR4, AVPR1B, and DCLK3), as well as numerous proteins with evidence of existence on the protein level that previously lacked information on spatial resolution and cell-type- specific expression pattern. We here present a comprehensive strategy for identification of missing proteins by combining transcriptomics with antibody-based proteomics. The analyzed proteins provide interesting targets for organ-specific research in health and disease.
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| 2. |
- Edfors, Fredrik, et al.
(författare)
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Enhanced validation of antibodies for research applications
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.
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| 3. |
- Gremel, Gabriela, et al.
(författare)
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A systematic search strategy identifies cubilin as independent prognostic marker for renal cell carcinoma
- 2017
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is an unmet clinical need for better prognostic and diagnostic tools for renal cell carcinoma (RCC). Methods: Human Protein Atlas data resources, including the transcriptomes and proteomes of normal and malignant human tissues, were searched for RCC-specific proteins and cubilin (CUBN) identified as a candidate. Patient tissue representing various cancer types was constructed into a tissue microarray (n = 940) and immunohistochemistry used to investigate the specificity of CUBN expression in RCC as compared to other cancers. Two independent RCC cohorts (n = 181; n = 114) were analyzed to further establish the sensitivity of CUBN as RCC-specific marker and to explore if the fraction of RCCs lacking CUBN expression could predict differences in patient survival. Results: CUBN was identified as highly RCC-specific protein with 58% of all primary RCCs staining positive for CUBN using immunohistochemistry. In venous tumor thrombi and metastatic lesions, the frequency of CUBN expression was increasingly lost. Clear cell RCC (ccRCC) patients with CUBN positive tumors had a significantly better prognosis compared to patients with CUBN negative tumors, independent of T-stage, Fuhrman grade and nodal status (HR 0.382, CI 0.203-0.719, P = 0.003). Conclusions: CUBN expression is highly specific to RCC and loss of the protein is significantly and independently associated with poor prognosis. CUBN expression in ccRCC provides a promising positive prognostic indicator for patients with ccRCC. The high specificity of CUBN expression in RCC also suggests a role as a new diagnostic marker in clinical cancer differential diagnostics to confirm or rule out RCC.
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| 4. |
- Jennbacken, Karin, et al.
(författare)
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Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 20:23
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Tidskriftsartikel (refereegranskat)abstract
- Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naive cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.
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| 5. |
- Lundqvist, Magnus, et al.
(författare)
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Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:7
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Tidskriftsartikel (refereegranskat)abstract
- We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.
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| 6. |
- Thul, Peter J., et al.
(författare)
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A subcellular map of the human proteome
- 2017
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Ingår i: Science. - : American Association for the Advancement of Science. - 0036-8075 .- 1095-9203. ; 356:6340
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Tidskriftsartikel (refereegranskat)abstract
- Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.
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| 7. |
- Uhlén, Mathias, et al.
(författare)
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A genome-wide transcriptomic analysis of protein-coding genes in human blood cells
- 2019
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Ingår i: Science. - : American Association for the Advancement of Science. - 0036-8075 .- 1095-9203. ; 366:6472, s. 1471-
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Tidskriftsartikel (refereegranskat)abstract
- Blood is the predominant source for molecular analyses in humans, both in clinical and research settings. It is the target for many therapeutic strategies, emphasizing the need for comprehensive molecular maps of the cells constituting human blood. In this study, we performed a genome-wide transcriptomic analysis of protein-coding genes in sorted blood immune cell populations to characterize the expression levels of each individual gene across the blood cell types. All data are presented in an interactive, open-access Blood Atlas as part of the Human Protein Atlas and are integrated with expression profiles across all major tissues to provide spatial classification of all protein-coding genes. This allows for a genome-wide exploration of the expression profiles across human immune cell populations and all major human tissues and organs.
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| 8. |
- Uhlén, Mathias, et al.
(författare)
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The human secretome
- 2019
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 12:609
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.
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| 9. |
- Uhlén, Mathias, et al.
(författare)
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Tissue-based map of the human proteome
- 2015
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 347:6220, s. 1260419-
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Tidskriftsartikel (refereegranskat)abstract
- Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
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