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Profiling cellular protein complexes by proximity ligation with dual tag microarray readout
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, e40405- p.Article in journal (Refereed) Published
Abstract [en]

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

Place, publisher, year, edition, pages
2012. Vol. 7, no 7, e40405- p.
Keyword [en]
proximity ligation assay, protein interaction, dual tag microarray
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-177984DOI: 10.1371/journal.pone.0040405ISI: 000306355500039PubMedID: 22808155OAI: oai:DiVA.org:uu-177984DiVA: diva2:541726
Funder
Swedish Research CouncilNIH (National Institute of Health), 1R21CA126727-01A1
Note

De 2 första författarna delar förstaförfattarskapet.

Correction in: PLoS ONE 10(3): e0119890. doi: 10.1371/journal.pone.0119890ISI: 000351880000047

Available from: 2012-07-23 Created: 2012-07-23 Last updated: 2017-12-07Bibliographically approved
In thesis
1. DNA-Mediated Detection and Profiling of Protein Complexes
Open this publication in new window or tab >>DNA-Mediated Detection and Profiling of Protein Complexes
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules.

Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 923
Keyword
Proximity ligation assay, Protein complexes, Protein interactions, Biomarkers, Prions, Antibodies
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-204861 (URN)978-91-554-8718-8 (ISBN)
Public defence
2013-09-27, Rudbecksalen, Rudbeck Laboratory, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2013-09-06 Created: 2013-08-12 Last updated: 2014-01-08
2. Proximity Ligation Assays for Disease Biomarkers Analysis
Open this publication in new window or tab >>Proximity Ligation Assays for Disease Biomarkers Analysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

One of the pressing needs in the field of disease biomarker discovery is new technologies that could allow high performance protein analysis in different types of clinical material, such as blood and solid tissues. This thesis includes four approaches that address important limitations of current technologies, thus enabling highly sensitive, specific and parallel protein measurements.

Paper I describes a method for sensitive singleplex protein detection in complex biological samples, namely solid phase proximity ligation assay (SP-PLA). SP-PLA exhibited improved sensitivity compared to conventional sandwich immunoassays. We applied SP-PLA to validate the potential of GDF-15 as a biomarker for cardiovascular disease.  

Paper II describes ProteinSeq, a multiplexed immunoassay based on the principle of SP-PLA, for parallel detection of 36 proteins using next-generation sequencing as readout. ProteinSeq exhibited improved sensitivity compared to multiplexed sandwich immunoassays, and the potential to achieve even higher levels of multiplexing while preserving a high sensitivity and specificity. We applied ProteinSeq to analyze 36 proteins, including one internal control, in 5 μl of plasma samples in a cohort of patients with cardiovascular disease and healthy controls.

Paper III describes PLA-DTM, a strategy for recording all possible interactions between sets of proteins in clinical samples. Individual proteins and their interactions are first encoded to dual barcoded DNA by PLA, and the barcodes are interrogated by a method named dual tag microarray (DTM). We applied the method for studying interactions among protein members of the NFκB signaling pathway.

Paper IV describes a novel probing strategy for analyzing individual biomolecules in solution or in situ. The technique employs a new class of probes for unfolding proximity ligation assays - uPLA probes. The probes are designed so that each probe set is sufficient in forming and replicating circular DNA reporter, without interactions among themselves when incubated with the sample. The uPLA probing strategy provides ease in the design of multiple probe sets in parallelized assays while enhancing the specificity of detection. We used the uPLA probes to detect various targets, including synthetic DNA and cancer-related transcripts in situ.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 703
Keyword
proximity ligation assay, blood biomarkers, protein interactions, pathway analysis, single molecule, next-generation sequencing
National Category
Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-158634 (URN)978-91-554-8158-2 (ISBN)
Public defence
2011-10-28, Rudbecksalen, Rudbecklaboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Supervisors
Available from: 2011-10-07 Created: 2011-09-12 Last updated: 2015-08-10Bibliographically approved

Open Access in DiVA

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