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Antibody-based protein profiling of the human chromosome 21.

Uhlén M, Oksvold P, Älgenäs C, Hamsten C, Fagerberg L, Klevebring D, Lundberg E, Odeberg J, Pontén F, Kondo T, Sivertsson Å - Mol. Cell Proteomics (2011)

Bottom Line: We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing.The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level, and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms.The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Royal Institute of Technology, SE-17121 Solna, Sweden. mathias.uhlen@scilifelab.se

ABSTRACT
The Human Proteome Project has been proposed to create a knowledge-based resource based on a systematical mapping of all human proteins, chromosome by chromosome, in a gene-centric manner. With this background, we here describe the systematic analysis of chromosome 21 using an antibody-based approach for protein profiling using both confocal microscopy and immunohistochemistry, complemented with transcript profiling using next generation sequencing data. We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing. The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level, and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms. A chromosome-wide matrix is presented with status for all chromosome 21 genes regarding subcellular localization, tissue distribution, and molecular characterization of the corresponding proteins. The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.

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Overview of data for proteins on chromosome 21. A status matrix is shown where, for each gene, the evidence levels according to UniProt (first columns), the current status in the Human Protein Atlas (second columns), the result of molecular characterization using Western blot (third columns), the reliability of the results from subcellular location analysis (fourth columns), and tissue profiling (fifth columns), and the evidence for existence on transcriptional level in cell lines (sixth columns) are represented using four colors as follows. First columns: green, protein evidence; yellow, transcript evidence; red, uncertain; black, not reviewed. Second columns: green, at least one antibody approved by the Human Protein Atlas; yellow, MS-verified antigen generated; red, failed or not started; black, in progress. Third through sixth columns: green, supportive; yellow, uncertain; red, nonsupportive; black, not done.
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Figure 5: Overview of data for proteins on chromosome 21. A status matrix is shown where, for each gene, the evidence levels according to UniProt (first columns), the current status in the Human Protein Atlas (second columns), the result of molecular characterization using Western blot (third columns), the reliability of the results from subcellular location analysis (fourth columns), and tissue profiling (fifth columns), and the evidence for existence on transcriptional level in cell lines (sixth columns) are represented using four colors as follows. First columns: green, protein evidence; yellow, transcript evidence; red, uncertain; black, not reviewed. Second columns: green, at least one antibody approved by the Human Protein Atlas; yellow, MS-verified antigen generated; red, failed or not started; black, in progress. Third through sixth columns: green, supportive; yellow, uncertain; red, nonsupportive; black, not done.

Mentions: In Fig. 5, a detailed matrix is shown with the status of the experimental characterization of the proteins encoded by chromosome 21 genes, here excluding the keratin-associated proteins, with further details presented in supplemental Table 3. The first column shows the status of the annotation performed by Uniprot for the 192 putative genes with the color code according to Fig. 1. The second column shows the status of the generation of antibodies with a green box representing genes with at least one antibody approved by the Human Protein Atlas program and available to the public. Most of the remaining genes have a yellow color code, indicating that at least one antigen corresponding to a unique region of the corresponding protein target has been expressed, purified, and verified by mass spectrometry as part of the Human Protein Atlas program. At present, only four putative genes have failed attempts to generate antigens: APOO1346.1, AFO15262.1, DSCR8, and C21orf33. Two of these lack transcript evidence according to UniProt, and corresponding transcripts have not been detected in any of the three diverse cell lines assayed using RNA-seq, which calls for more in-depth studies to confirm the annotation of these putative genes as protein coding.


Antibody-based protein profiling of the human chromosome 21.

Uhlén M, Oksvold P, Älgenäs C, Hamsten C, Fagerberg L, Klevebring D, Lundberg E, Odeberg J, Pontén F, Kondo T, Sivertsson Å - Mol. Cell Proteomics (2011)

Overview of data for proteins on chromosome 21. A status matrix is shown where, for each gene, the evidence levels according to UniProt (first columns), the current status in the Human Protein Atlas (second columns), the result of molecular characterization using Western blot (third columns), the reliability of the results from subcellular location analysis (fourth columns), and tissue profiling (fifth columns), and the evidence for existence on transcriptional level in cell lines (sixth columns) are represented using four colors as follows. First columns: green, protein evidence; yellow, transcript evidence; red, uncertain; black, not reviewed. Second columns: green, at least one antibody approved by the Human Protein Atlas; yellow, MS-verified antigen generated; red, failed or not started; black, in progress. Third through sixth columns: green, supportive; yellow, uncertain; red, nonsupportive; black, not done.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316724&req=5

Figure 5: Overview of data for proteins on chromosome 21. A status matrix is shown where, for each gene, the evidence levels according to UniProt (first columns), the current status in the Human Protein Atlas (second columns), the result of molecular characterization using Western blot (third columns), the reliability of the results from subcellular location analysis (fourth columns), and tissue profiling (fifth columns), and the evidence for existence on transcriptional level in cell lines (sixth columns) are represented using four colors as follows. First columns: green, protein evidence; yellow, transcript evidence; red, uncertain; black, not reviewed. Second columns: green, at least one antibody approved by the Human Protein Atlas; yellow, MS-verified antigen generated; red, failed or not started; black, in progress. Third through sixth columns: green, supportive; yellow, uncertain; red, nonsupportive; black, not done.
Mentions: In Fig. 5, a detailed matrix is shown with the status of the experimental characterization of the proteins encoded by chromosome 21 genes, here excluding the keratin-associated proteins, with further details presented in supplemental Table 3. The first column shows the status of the annotation performed by Uniprot for the 192 putative genes with the color code according to Fig. 1. The second column shows the status of the generation of antibodies with a green box representing genes with at least one antibody approved by the Human Protein Atlas program and available to the public. Most of the remaining genes have a yellow color code, indicating that at least one antigen corresponding to a unique region of the corresponding protein target has been expressed, purified, and verified by mass spectrometry as part of the Human Protein Atlas program. At present, only four putative genes have failed attempts to generate antigens: APOO1346.1, AFO15262.1, DSCR8, and C21orf33. Two of these lack transcript evidence according to UniProt, and corresponding transcripts have not been detected in any of the three diverse cell lines assayed using RNA-seq, which calls for more in-depth studies to confirm the annotation of these putative genes as protein coding.

Bottom Line: We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing.The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level, and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms.The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Royal Institute of Technology, SE-17121 Solna, Sweden. mathias.uhlen@scilifelab.se

ABSTRACT
The Human Proteome Project has been proposed to create a knowledge-based resource based on a systematical mapping of all human proteins, chromosome by chromosome, in a gene-centric manner. With this background, we here describe the systematic analysis of chromosome 21 using an antibody-based approach for protein profiling using both confocal microscopy and immunohistochemistry, complemented with transcript profiling using next generation sequencing data. We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing. The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level, and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms. A chromosome-wide matrix is presented with status for all chromosome 21 genes regarding subcellular localization, tissue distribution, and molecular characterization of the corresponding proteins. The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.

Show MeSH
Related in: MedlinePlus