File Download
Supplementary

postgraduate thesis: Fluorescence probes based on NTA-metal coordination chemistry for metalloproteomics

TitleFluorescence probes based on NTA-metal coordination chemistry for metalloproteomics
Authors
Advisors
Advisor(s):Sun, H
Issue Date2018
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Jiang, N. [江南]. (2018). Fluorescence probes based on NTA-metal coordination chemistry for metalloproteomics. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractA dynamic balance of metals is absolutely indispensable for all types of lives whilst the importance of metals comes from the crucial roles of metalloproteins in life processes. Detections of intracellular metals and metalloproteins have been achieved by multiple approaches including mass spectra-based techniques, X-ray fluorescence with total reflection and fluorescence sensors. Taking the benefits of real-time visualization in living cells, fluorescence-based sensors have been widely explored and applied to the exploration of metallic biology. Given the versatility of nitrilotriacetate (NTA) in metal-binding, a family of NTA-metal based fluorescence probes was designed and synthesized to track intracellular metalloproteins via metal coordination in biological systems. Metal-associated proteomes were uncovered in the pathogens, mammalian cells and tissues. On one hand, proteomics and bioinformatics were then integrated into the imaging of the probe, Fe-TRACER, for mining the iron-associated proteome. 17 iron-associated proteins were identified in Porphyromonas gingivalis, which acts as a keystone oral pathogen in periodontitis and threatens the dental health of 10–15% adults worldwide. A similar Cu-associated proteome tracking approach was applied to the case of Alzheimer disease with the combination of labile Cu(I) pool imaging and total copper content measurement. The Cu-associated changes in this disease were analysed as well as the Cu-associated proteins were mined for the potential targets of therapeutic strategies. Simultaneously, considering the wide usage of His6-Ni-NTA system in protein purification and biotechnology, this family of fluorescence probes was employed to label intracellular His-tagged proteins. Inheriting the great success of the former study of Ni-NTA-AC, a novel red fluorescence probe, Ni-NTA-AB, was synthesized and it is composed of the Ni-NTA moiety and a BODIPY fluorophore connected with an arylazide group. This probe molecule entered living E. coli cells to light up the His-tagged proteins with the assistance of Tween 80. The labelled proteins can be quantified using SDS-PAGE by calculating the fluorescence intensity in a wider linear range (25–1000 ng) than that of Western Blot. The development of the Raman probe in metabonomics led me to replace the fluorophore with alkynyl group, as the limitation in the enlargement of fluorophores was realized. Herein, the NTA group was covalently connected to alkynyl group and the subcellular locations of the new metal complexes were demonstrated to be determined by metal-binding in the stimulated Raman scattering imaging. A probe family based upon metal-NTA coordination chemistry was designed and built whilst this kind of probes can covalently label intracellular metal-associated proteins for the subsequent identification, quantification and analysis. This may provide a powerful toolbox to explore metalloproteins and metalloproteomes in diverse biological systems.
DegreeDoctor of Philosophy
SubjectMetalloproteins
Fluorescent probes
Coordination compounds
Dept/ProgramChemistry
Persistent Identifierhttp://hdl.handle.net/10722/268437

 

DC FieldValueLanguage
dc.contributor.advisorSun, H-
dc.contributor.authorJiang, Nan-
dc.contributor.author江南-
dc.date.accessioned2019-03-21T01:40:24Z-
dc.date.available2019-03-21T01:40:24Z-
dc.date.issued2018-
dc.identifier.citationJiang, N. [江南]. (2018). Fluorescence probes based on NTA-metal coordination chemistry for metalloproteomics. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/268437-
dc.description.abstractA dynamic balance of metals is absolutely indispensable for all types of lives whilst the importance of metals comes from the crucial roles of metalloproteins in life processes. Detections of intracellular metals and metalloproteins have been achieved by multiple approaches including mass spectra-based techniques, X-ray fluorescence with total reflection and fluorescence sensors. Taking the benefits of real-time visualization in living cells, fluorescence-based sensors have been widely explored and applied to the exploration of metallic biology. Given the versatility of nitrilotriacetate (NTA) in metal-binding, a family of NTA-metal based fluorescence probes was designed and synthesized to track intracellular metalloproteins via metal coordination in biological systems. Metal-associated proteomes were uncovered in the pathogens, mammalian cells and tissues. On one hand, proteomics and bioinformatics were then integrated into the imaging of the probe, Fe-TRACER, for mining the iron-associated proteome. 17 iron-associated proteins were identified in Porphyromonas gingivalis, which acts as a keystone oral pathogen in periodontitis and threatens the dental health of 10–15% adults worldwide. A similar Cu-associated proteome tracking approach was applied to the case of Alzheimer disease with the combination of labile Cu(I) pool imaging and total copper content measurement. The Cu-associated changes in this disease were analysed as well as the Cu-associated proteins were mined for the potential targets of therapeutic strategies. Simultaneously, considering the wide usage of His6-Ni-NTA system in protein purification and biotechnology, this family of fluorescence probes was employed to label intracellular His-tagged proteins. Inheriting the great success of the former study of Ni-NTA-AC, a novel red fluorescence probe, Ni-NTA-AB, was synthesized and it is composed of the Ni-NTA moiety and a BODIPY fluorophore connected with an arylazide group. This probe molecule entered living E. coli cells to light up the His-tagged proteins with the assistance of Tween 80. The labelled proteins can be quantified using SDS-PAGE by calculating the fluorescence intensity in a wider linear range (25–1000 ng) than that of Western Blot. The development of the Raman probe in metabonomics led me to replace the fluorophore with alkynyl group, as the limitation in the enlargement of fluorophores was realized. Herein, the NTA group was covalently connected to alkynyl group and the subcellular locations of the new metal complexes were demonstrated to be determined by metal-binding in the stimulated Raman scattering imaging. A probe family based upon metal-NTA coordination chemistry was designed and built whilst this kind of probes can covalently label intracellular metal-associated proteins for the subsequent identification, quantification and analysis. This may provide a powerful toolbox to explore metalloproteins and metalloproteomes in diverse biological systems. -
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshMetalloproteins-
dc.subject.lcshFluorescent probes-
dc.subject.lcshCoordination compounds-
dc.titleFluorescence probes based on NTA-metal coordination chemistry for metalloproteomics-
dc.typePG_Thesis-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineChemistry-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2019-
dc.identifier.mmsid991044091306403414-

Export via OAI-PMH Interface in XML Formats


OR


Export to Other Non-XML Formats