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Affinity based proteomics research tools
KTH, School of Biotechnology (BIO), Proteomics.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Listen to the mantra; the mapping of the genome was finished in 2001, and the sequel research challenge is the thorough survey of the corresponding human proteome. This was stated almost a decade ago, it has been repeated over and over, and is still most certainly a hard nut to crack. The workload is daunting, much because there is no protein amplification method and no binary system for the detection of proteins, and because the complexity of the proteome is larger than that of the genome as it seems. Hence, there is a need for high throughput technologies that, at sufficiently low limits of detection and with satisfying sensitivities, may investigate protein content in human samples. With this aim, the Human Proteome Resource (HPR) project was initiated in 2003.

All work presented in this thesis relate to protein interactions; binders are either utilized such as for the depletion of high abundant proteins from serum, or analyzed such as in the validation of monospecific antibody specificity, or the epitope mapping of the same. In Paper I, the Gyrolab system is utilized in a setup for the specificity analysis of monospecific antibodies towards their antigen, and the setup is compared to planar protein arrays. Gyrolab technology is used again, in Paper II, where epitope mapping of monospecific antibodies is performed in order to analyze antibody specificity. Also, mapping serves to compare the immune-responses from parallel immunizations using the same antigen, thereby assessing reproducibility in the regeneration of antibodies. Paper III describes a high throughput approach for the depletion of high abundant proteins, in serum and plasma samples, taking advantage of Affibody molecules as binders. The last two papers, IV and V, utilize monospecific antibodies for protein analysis; in Paper IV pull out experiments show that competitive elution using the PrEST antigen can be a fruitful approach to increase specificity. And finally, in Paper V, a setup for the semi-quantitative protein content analysis in fluid samples is suggested. Again, the Gyrolab technology is used, and the setup is tested on a simplified model system.

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , 86 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:15
National Category
Immunology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-11184ISBN: 978-91-7415-422-1 (print)OAI: oai:DiVA.org:kth-11184DiVA: diva2:241104
Public defence
2009-10-09, FR4 Oskar Klein, AlbaNova, Roslagstullsbacken 33, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100712Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2010-07-16Bibliographically approved
List of papers
1. Microfluidic analysis of antibody specificity in a compact disk format
Open this publication in new window or tab >>Microfluidic analysis of antibody specificity in a compact disk format
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2006 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 7, 1568-1574 p.Article in journal (Refereed) Published
Abstract [en]

A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His(6)-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three- dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.

Keyword
microfluidics, miniaturization, compact disk, biochip, gyrolab, antibody specificity, IMMUNOSORBENT-ASSAY ELISA, PROTEOMICS, MICROARRAYS, GENOME, CHIP
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14074 (URN)10.1021/pr050447c (DOI)000238838500007 ()2-s2.0-33745862053 (Scopus ID)
Note
QC 20100712Available from: 2010-07-12 Created: 2010-07-12 Last updated: 2017-12-12Bibliographically approved
2. Characterization of PrEST-based antibodies towards human Cytokeratin-17
Open this publication in new window or tab >>Characterization of PrEST-based antibodies towards human Cytokeratin-17
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2009 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 342, 20-32 p.Article in journal (Refereed) Published
Abstract [en]

Antibody-based proteomics efforts depend on validated antibodies to ensure correct annotation of analyzed proteins. We have previously argued that a low sequence identity to other proteins is a key feature for antigens used in antibody generation. Thus, a major challenge for whole-proteome studies is how to address families of highly sequence related proteins within the context of generating specific antibodies. In this study, two non-overlapping parts of human Cytokeratin-17, a protein belonging to the intermediate filament family of highly sequence-related proteins, were selected as a model system to study the specificity and cross reactivity of antibodies generated towards such a target. These recombinantly produced Protein Epitope Signature Tags (PrESTs) were immunized in five rabbits each and the batch-to-batch variations in the obtained immune responses were studied by mapping of linear epitopes using synthetic overlapping peptides. The obtained results showed a similar but not identical immune response in the respective antibody groups with a limited number of epitopes being identified. Immunohistochemical analysis of the affinity purified monospecific antibodies on tissue micro arrays resulted in a general recognition of human cytokeratins for all analyzed binders whereas antibodies identified as binding to the most unique parts of the PrESTs showed the most Cytokeratin-17 like staining. The data presented here support the strategy to use sequence identity scores as the main criteria for antigen selection but also indicate the possibility to instead produce a single antibody recognizing a defined group of proteins when the intended targets overall sequence identity score is too high. This type of group-specific antibodies would be an important tool for antibody-based projects aiming for a complete coverage of the human proteome.

Keyword
Cytokeratin-17; PrEST; Epitope mapping; Antibody
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11430 (URN)10.1016/j.jim.2008.11.013 (DOI)000264685300003 ()
Note
QC 20100712Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2017-12-12Bibliographically approved
3. Affibody molecule mediated depletion of HSA and IgG performed in singlet or in a rapid high throughput format
Open this publication in new window or tab >>Affibody molecule mediated depletion of HSA and IgG performed in singlet or in a rapid high throughput format
2009 (English)Article in journal (Other academic) Submitted
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14076 (URN)
Note
QS 20120315Available from: 2010-07-12 Created: 2010-07-12 Last updated: 2012-03-15Bibliographically approved
4. Targeted protein pullout from human tissue samples using competitive elution
Open this publication in new window or tab >>Targeted protein pullout from human tissue samples using competitive elution
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2011 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 6, no 1, 28-37 p.Article in journal (Refereed) Published
Abstract [en]

One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, I MAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.

Keyword
Antibody, Immunoprecipitation, Methods, Proteomics, Pullout
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14079 (URN)10.1002/biot.201000341 (DOI)000287716300002 ()2-s2.0-78651306981 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20100712 Uppdaterad från manuskript till artikel (20110316).Available from: 2010-07-12 Created: 2010-07-12 Last updated: 2011-12-07Bibliographically approved
5. Towards an immunobased tool for the qualification of bookmarker candidates-compact disk enclosed nanocolumns with orientation-immobilized monospecific antibodies.
Open this publication in new window or tab >>Towards an immunobased tool for the qualification of bookmarker candidates-compact disk enclosed nanocolumns with orientation-immobilized monospecific antibodies.
(English)Article in journal (Other academic) Submitted
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14082 (URN)
Note
QS 20120326Available from: 2010-07-12 Created: 2010-07-12 Last updated: 2012-03-26Bibliographically approved

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