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Profiling individual protein complexes by proximity-dependent barcoding
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.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-220093OAI: oai:DiVA.org:uu-220093DiVA: diva2:704286
Available from: 2014-03-11 Created: 2014-03-10 Last updated: 2015-02-03
In thesis
1. Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
Open this publication in new window or tab >>Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed.

In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences.

In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA).

In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls.

In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS.

In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 980
Keyword
proximity ligation assay, proximity barcoding assay, sensitive, in situ, multiplex, profiling, amyloid-beta, MARK, tau, CSF, biomarker, protein complexes, Alzheimer’s disease, amyotrophic lateral sclerosis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-220070 (URN)978-91-554-8901-4 (ISBN)
Public defence
2014-04-25, B21, Biomedicinskt centrum, Husargatan 3, SE-75123, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2014-04-03 Created: 2014-03-10 Last updated: 2014-04-29
2. DNA-Assisted Immunoassays for High-Performance Protein Analyses
Open this publication in new window or tab >>DNA-Assisted Immunoassays for High-Performance Protein Analyses
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages.

In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1055
Keyword
proximity ligation assay, magnetic beads, conjugation, phosphorylation, affinity binder, protein complexes, recombinant binder, sensitivity, specificity
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-236591 (URN)978-91-554-9114-7 (ISBN)
Public defence
2015-01-16, B41, Biomedicalcenter, Husargatan 3, 75018, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-12-19 Created: 2014-11-20 Last updated: 2015-02-03

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