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ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing
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. (Ulf Landegren)
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. (Ulf Landegren)
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. (Ulf Landegren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, e25583- p.Article in journal (Refereed) Published
Abstract [en]

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 μl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use. 

Place, publisher, year, edition, pages
2011. Vol. 6, no 9, e25583- p.
Keyword [en]
proximity ligation assay, next generation sequencing, cardiovascular disease, biomarker
National Category
Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-158802DOI: 10.1371/journal.pone.0025583ISI: 000295939600036PubMedID: 21980495OAI: oai:DiVA.org:uu-158802DiVA: diva2:441209
Available from: 2011-09-15 Created: 2011-09-15 Last updated: 2017-12-08Bibliographically approved
In thesis
1. 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

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Nong, Rachel YuanVänelid, JohanSiegbahn, AgnetaBäcklin, ChristoferWallentin, LarsGustafsson, Mats G.Kamali-Moghaddam, MasoodLandegren, Ulf

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Nong, Rachel YuanVänelid, JohanSiegbahn, AgnetaBäcklin, ChristoferWallentin, LarsGustafsson, Mats G.Kamali-Moghaddam, MasoodLandegren, Ulf
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Molecular toolsScience for Life Laboratory, SciLifeLabClinical ChemistryDepartment of Medical SciencesUCR-Uppsala Clinical Research Center
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