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Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
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 [en]
Mass spectrometry, proteomics, microarray, protein, antibody, antigen, affinity, validation
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-259623ISBN: 978-91-554-9300-4 (print)OAI: oai:DiVA.org:uu-259623DiVA: diva2:845074
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
List of papers
1. Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling
Open this publication in new window or tab >>Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling
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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.

Keyword
Antibody generation, protein microarray, proteome atlas, tissue microarray, tissue profiling
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-80602 (URN)10.1002/pmic.200500072 (DOI)16237735 (PubMedID)
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
2. 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:uu:diva-208954 (URN)10.1021/pr050447c (DOI)000238838500007 ()
Note

QC 20100712

Available from: 2010-07-12 Created: 2013-10-11 Last updated: 2017-12-06
3. Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
Open this publication in new window or tab >>Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
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2007 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 1, 125-132 p.Article in journal (Refereed) Published
Abstract [en]

One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.

Keyword
protein microarrays, atlas
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-208952 (URN)10.1074/mcp.T60035-MCP200 (DOI)000243312000011 ()
Note

QC 20100525

Available from: 2010-08-05 Created: 2013-10-11 Last updated: 2017-12-06
4. Validation of affinity reagents using antigen microarrays
Open this publication in new window or tab >>Validation of affinity reagents using antigen microarrays
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2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, 555-563 p.Article in journal (Other academic) Published
Abstract [en]

There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

Keyword
protein microarray, antibody validation, affinity reagent, antigen, specificity, SH2
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-208949 (URN)10.1016/j.nbt.2011.11.009 (DOI)000305606500007 ()
Available from: 2011-11-17 Created: 2013-10-11 Last updated: 2017-12-06
5. Estrogen receptor beta profiling in human tissues following extensive antibody validation
Open this publication in new window or tab >>Estrogen receptor beta profiling in human tissues following extensive antibody validation
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(English)Manuscript (preprint) (Other academic)
Keyword
Estrogen receptor beta, ER-beta, IHC, antibody validation, TMA
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-251340 (URN)
Available from: 2015-04-21 Created: 2015-04-15 Last updated: 2015-10-01
6. Quantitative and selective analysis of feline growth related proteins using parallel reaction monitoring high resolution mass spectrometry
Open this publication in new window or tab >>Quantitative and selective analysis of feline growth related proteins using parallel reaction monitoring high resolution mass spectrometry
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(English)Manuscript (preprint) (Other academic)
Keyword
Parallel reaction monitoring, QPrEST, targeted proteomics, cat, feline, Orbitrap, mass spectrometry, IGF-I, IGF-II, IGFBP-3, IGFBP-5
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-259614 (URN)
Available from: 2015-08-10 Created: 2015-08-10 Last updated: 2015-10-01
7. High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry
Open this publication in new window or tab >>High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry
2015 (English)In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 3, 68-75 p.Article in journal (Refereed) Published
Abstract [en]

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals. In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.

Keyword
Cerebrospinal fluid, Dog, High-abundant protein depletion, Shotgun proteomics, Orbitrap, Mass spectrometry
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-259611 (URN)10.1016/j.bbrep.2015.07.013 (DOI)
Available from: 2015-08-10 Created: 2015-08-10 Last updated: 2015-10-01Bibliographically approved

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