| 1. |
- Berglund, Lisa, et al.
(författare)
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Generation of validated antibodies towards the human proteome
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Here we show the results from a large effort to generate antibodies towards the human proteome. A high-throughput strategy was developed based on cloning and expression of antigens as recombitant protein epitope signature tags (PrESTs) Affinity purified polyclonal antibodies were generated, followed by validation by protein microarrays, Western blotting and microarray-based immunohistochemistry. PrESTs were selected based on sequence uniqueness relative the proteome and a bioinformatics analysis showed that unique antigens can be found for at least 85% of the proteome using this general strategy. The success rate from antigen selection to validated antibodies was 31%, and from protein to antibody 55%. Interestingly, membrane-bound and soluble proteins performed equally and PrEST lengths between 75 and 125 amino acids were found to give the highest yield of validated antibodies. Multiple antigens were selected for many genes and the results suggest that specific antibodies can be systematically generated to most human proteibs.
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| 2. |
- Fagerberg, Linn, et al.
(författare)
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Large-Scale Protein Profiling in Human Cell Lines Using Antibody-Based Proteomics
- 2011
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 10:9, s. 4066-4075
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Tidskriftsartikel (refereegranskat)abstract
- Human cancer cell lines grown in vitro are frequently used to decipher basic cell biological phenomena and to also specifically study different forms of cancer. Here we present the first large-scale study of protein expression patterns in cell lines using an antibody-based proteomics approach. We analyzed the expression pattern of 5436 proteins in 45 different cell lines using hierarchical clustering, principal component analysis, and two-group comparisons for the identification of differentially expressed proteins. Our results show that immunohistochemically determined protein profiles can categorize cell lines into groups that overall reflect the tumor tissue of origin and that hematological cell lines appear to retain their protein profiles to a higher degree than cell lines established from solid tumors. The two-group comparisons reveal well-characterized proteins as well as previously unstudied proteins that could be of potential interest for further investigations. Moreover, multiple myeloma cells and cells of myeloid origin were found to share a protein profile, relative to the protein profile of lymphoid leukemia and lymphoma cells, possibly reflecting their common dependency of bone marrow microenvironment. This work also provides an extensive list of antibodies, for which high-resolution images as well as validation data are available on the Human Protein Atlas (www.proteinatlas.org), that are of potential use in cell line studies.
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| 3. |
- Fagerberg, Linn, et al.
(författare)
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The Global Protein Expression Pattern in Human Cell Lines
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Human cancer cell lines grown in vitro are frequently used to decipher basic cell biological phenomena but also to specifically study different forms of cancer. Here we present the first large-scale study of protein expression patterns in cell lines using an antibody-based proteomics approach. We analyzed the expression pattern of 5436 proteins in 45 different cell lines using hierarchical clustering, principal component analysis and two-group comparisons for the identification of differentially expressed proteins. The results show that protein profiles of cell lines, as determined using immunohistochemistry, allow for a hierarchical clustering that overall reflects tumor tissues of origin. Hematological cell lines appear to retain their protein profiles to a higher degree than cell lines established from solid tumors, resulting in a clustering that well reflects progenitor cell types. The discrepancy may reflect different levels of in vitro induced alterations in adherent and suspension grown cell lines, respectively. In addition, multiple myeloma cells and cells of myeloid origin were found to share a protein profile, relative the protein profile of lymphoid leukemia and lymphoma cells, possibly reflecting their common dependency of bone marrow microenvironment.
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| 4. |
- Gry, Marcus, et al.
(författare)
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Correlations between RNA and protein expression profiles in 23 human cell lines
- 2009
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays) and protein profiles (determined immunohistochemically in tissue microarrays) of 1066 gene products in 23 human cell lines. Results: A high mean correlation coefficient (0.52) was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Conclusion: Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies’ specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.
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| 5. |
- Gry, Marcus, et al.
(författare)
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Tissue-specific protein expression in human cells, tissues and organs
- 2010
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Ingår i: Journal of Proteomics and Bioinformatics. - : OMICS Publishing Group. - 0974-276X. ; 3:10, s. 286-293
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Tidskriftsartikel (refereegranskat)abstract
- An important part of understanding human biology is the study of tissue-specific expression both at the gene and protein level. In this study, the analysis of tissue specific protein expression was performed based on tissue micro array data available on the public Human Protein Atlas database (www.proteinatlas.org). An analysis of human proteins, corresponding to approximately one third of the protein-encoding genes, was carried out in 65 human tissues and cell types. The spatial distribution and relative abundance of 6,678 human proteins, were analyzed in different cell populations from various organs and tissues in the human body using unsupervised methods, such as hierarchical clustering and principal component analysis, as well as with supervised methods (Breiman, 2001). Well-known markers, such as neuromodulin for the central nervous system, keratin 20 for gastrointestinal tract and CD45 for hematopoietic cells, were identified as tissue-specific. Proteins expressed in a tissue-specific manner were identified for cells in all of the investigated tissues, including the central nervous system, hematopoietic system, squamous epithelium, mesenchymal cells and cells from the gastrointestinal tract. Several proteins not yet associated with tissue-specificity were identified, providing starting points for further studies to explore tissue-specific functions. This includes proteins with no known function, such as ZNF509 expressed in CNS and C1orf201 expressed in the gastro-intestinal tract. In general, the majority of the gene products are expressed in a ubiquitous manner and few proteins are detected exclusively in cells from a particular tissue class, as exemplified by less than 1% of the analyzed proteins found only in the brain.
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| 6. |
- Lundberg, Emma, et al.
(författare)
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The correlation between cellular size and protein expression levels--normalization for global protein profiling
- 2008
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Ingår i: Journal of proteomics. - : Elsevier BV. - 1874-3919. ; 71:4, s. 448-460
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Tidskriftsartikel (refereegranskat)abstract
- An automated image analysis system was used for protein quantification of 1862 human proteins in 47 cancer cell lines and 12 clinical cell samples using cell microarrays and immunohistochemistry. The analysis suggests that most proteins are expressed in a cell size dependent manner, and that normalization is required for comparative protein quantification in order to correct for the inherent bias of cell size and systematic ambiguities associated with immunohistochemistry. Two reference standards were evaluated, and normalized protein expression values were found to allow for protein profiling across a panel of morphologically diverse cells, revealing putative patterns of over- and underexpression. Using this approach, proteins with stable expression as well as cell-line specific expression were identified. The results demonstrate the value of large-scale, automated proteome analysis using immunohistochemistry, in revealing functional correlations and establishing methods to interpret and mine proteomic data.
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| 7. |
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| 8. |
- Magnusson, Kristina, et al.
(författare)
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SATB2 in Combination With Cytokeratin 20 Identifies Over 95% of all Colorectal Carcinomas
- 2011
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Ingår i: American Journal of Surgical Pathology. - 0147-5185 .- 1532-0979. ; 35:7, s. 937-948
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Tidskriftsartikel (refereegranskat)abstract
- The special AT-rich sequence-binding protein 2 (SATB2), a nuclear matrix-associated transcription factor and epigenetic regulator, was identified as a tissue type-specific protein when screening protein expression patterns in human normal and cancer tissues using an antibody-based proteomics approach. In this respect, the SATB2 protein shows a selective pattern of expression and, within cells of epithelial lineages, SATB2 expression is restricted to glandular cells lining the lower gastrointestinal tract. The expression of SATB2 protein is primarily preserved in cancer cells of colorectal origin, indicating that SATB2 could function as a clinically useful diagnostic marker to distinguish colorectal cancer (CRC) from other types of cancer. The aim of this study was to further explore and validate the specific expression pattern of SATB2 as a clinical biomarker and to compare SATB2 with the well-known cytokeratin 20 (CK20). Immunohistochemistry was used to analyze the extent of SATB2 expression in tissue microarrays with tumors from 9 independent cohorts of patients with primary and metastatic CRCs (n = 1882). Our results show that SATB2 is a sensitive and highly specific marker for CRC with distinct positivity in 85% of all CRCs, and that SATB2 and/or CK20 was positive in 97% of CRCs. In conclusion, the specific expression of SATB2 in a large majority of CRCs suggests that SATB2 can be used as an important complementary tool for the differential diagnosis of carcinoma of unknown primary origin.
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| 9. |
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| 10. |
- Pontén, Fredrik, et al.
(författare)
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A global view of protein expression in human cells, tissues, and organs
- 2009
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Ingår i: Molecular Systems Biology. - : EMBO. - 1744-4292 .- 1744-4292. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry- based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced. Molecular Systems Biology 5: 337; published online 22 December 2009; doi:10.1038/msb.2009.93
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