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Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2005 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 17, 4327-4337 p.Article in journal (Refereed) Published
Abstract [en]

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

Place, publisher, year, edition, pages
2005. Vol. 5, no 17, 4327-4337 p.
Keyword [en]
Antibody generation, protein microarray, proteome atlas, tissue microarray, tissue profiling
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-80602DOI: 10.1002/pmic.200500072PubMedID: 16237735OAI: oai:DiVA.org:uu-80602DiVA: diva2:108516
Note

LP, KL och JÖ contributed equally to this work.

Available from: 2007-04-18 Created: 2007-04-18 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Validation of antibodies for protein profiling: A study using immunohistochemistry on tissue microarrays
Open this publication in new window or tab >>Validation of antibodies for protein profiling: A study using immunohistochemistry on tissue microarrays
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The field of proteomics has rapidly expanded due to the completion of the human genome sequence. This thesis validates affinity-purified monospecific antibodies of polyclonal origin, for protein profiling in a broad spectrum of normal tissues and cells. Validation of antibodies is crucial for development of reliable binders for target proteins and this thesis evaluates the generation and application of large sets of msAbs in different settings. MsAbs were generated towards recombinant Protein Epitope Signature Tag (PrEST) antigens using a stringent affinity-purification strategy, presented in the first study. The specificity of msAbs was studied using reverse phase protein arrays and immunohistochemistry (IHC), and results presented over 90% success rate in the protein array analysis. In IHC, 81% of the msAbs displayed apparent specific staining in normal tissues. MsAbs were also compared with commercial analogs (cAbs) using IHC and Western blot. Results presented similar outcome between msAbs and cAbs in both applications, although interpretation suggested more extensive IHC staining patterns with msAbs than with monoclonal analogs. For antibody validation, an approach called paired antibodies was presented and involved the generation of two msAbs towards non-overlapping epitopes on the same protein. Similarities in protein detection between paired antibodies were studied using three different antibody-based methods. Similar results were observed in several applications, indicating that this strategy can be a useful tool for studying known and unknown proteins. Given the reliability of msAbs, they were also applied in a study investigating the impact of tissue fixatives on protein detection. The study showed that different fixation mechanisms appeared to affect protein recognition by indicating that aldehyde-based fixation, e.g. induced by neutral buffered formalin, was preferred for tissues used in IHC and non-aldehyde based fixation was applicable for tissues used in protein extraction analysis and Western blotting.

Conclusively, validation results suggest that msAbs are reliable affinity binders that can be used as valuable tools for proteome-wide protein profiling in tissues and cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 474
Keyword
antibody validation, monospecific antibodies, protein profiling, immunohistochemistry, tissue microarrays, western blot, fixatives
National Category
Cell and Molecular Biology
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-107471 (URN)978-91-554-7588-8 (ISBN)
Public defence
2009-09-25, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsv 20, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-09-04 Created: 2009-08-12 Last updated: 2009-09-04Bibliographically approved
2. Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
Open this publication in new window or tab >>Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody.

Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity.

Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 82 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1272
Keyword
Mass spectrometry, proteomics, microarray, protein, antibody, antigen, affinity, validation
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-259623 (URN)978-91-554-9300-4 (ISBN)
Public defence
2015-09-25, C4:305, BMC, Husargatan 3, Uppsala, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2015-09-03 Created: 2015-08-10 Last updated: 2015-10-01

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Nilsson, PeterPaavilainen, LindaSundberg, MårtenKampf, CarolineWester, KennethPonten, Fredrik

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