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postgraduate thesis: Immunocapture laser ionization mass spectrometry and liquid chromatography mass spectrometry for protein marker analysis

TitleImmunocapture laser ionization mass spectrometry and liquid chromatography mass spectrometry for protein marker analysis
Authors
Advisors
Advisor(s):Ng, KMSun, H
Issue Date2018
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Cheung, Y. H. [張任丰]. (2018). Immunocapture laser ionization mass spectrometry and liquid chromatography mass spectrometry for protein marker analysis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractMonitoring of protein marker levels is invaluable in clinical applications, such as disease diagnosis and drug development. Matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS) is widely employed in protein marker analysis, but it suffers from poor sensitivity and limits of detection for high mass protein analysis. Here, an immunocapture laser ionization mass spectrometry (LI-MS) assay using gold nanoparticles (AuNPs) conjugated antibodies for indirect protein detection was developed. With three characteristic properties, including localized surface plasmon resonance, high atom density and facile surface chemistry, a AuNP as an ion reservoir can generate abundant Au cluster ions for signal amplification upon laser ionization, and conjugate with antibody for targeting specific proteins. In this study, human chorionic gonadotropin (hCG) was used for method development. Factors that affect antibody density on an antibody array and background signal, including silanization time, antibody printing condition, and blocking agent were investigated. Based on the experimental conditions optimized in the current study, the limit of detection for hCG was determined to be 0.38 fmol. Moreover, the study revealed that increasing the size of AuNPs can improve sensitivity, with 80 nm AuNPs having 14 times higher sensitivity than 20 nm AuNPs. The developed immunocapture LI-MS assay was then employed to detect PML-RARA fusion protein in NB4 cells, a 120 kDa biomarker for acute promyelocytic leukemia (APL) diagnosis, where its high mass makes it difficult to be detected using MALDI-MS. In the study, the limit of detection for PML-RARA was found to be 10,000 NB4 cells, outperforming conventional Western blot analysis. Furthermore, the feasibility of silver nanoparticle (AgNP) as an ion reservoir for signal amplification was explored, with three times higher sensitivity compared with AuNPs of equal particle size. The results demonstrated the capability of the immunocapture LI-MS assay in trace protein detection in biological samples with complex matrices. Finally, MS-based proteomics with tandem mass tags labelling was adopted to investigate the effect of arsenic trioxide (ATO) on protein expression levels in NB4 cells. In the study, 2,226 proteins were identified across three biological replicates. Pathway enrichment analysis conducted on 170 significantly upregulated proteins and 106 downregulated proteins revealed that spliceosome, carbon metabolism, and mRNA surveillance pathway and decay were upregulated, while separation of sister chromatids and protein processing in endoplasmic reticulum were downregulated upon ATO treatment. Furthermore, the study observed a two-fold decrease in PML level after ATO treatment, in accordance with the immunocapture LI-MS assay results. Therefore, MS-based proteomics combined with immunocapture LI-MS assay can constitute an attractive tool for cross validation in proteomic analysis.
DegreeMaster of Philosophy
SubjectHigh performance liquid chromatography
Mass spectrometry
Spectra - Proteins
Dept/ProgramChemistry
Persistent Identifierhttp://hdl.handle.net/10722/267761

 

DC FieldValueLanguage
dc.contributor.advisorNg, KM-
dc.contributor.advisorSun, H-
dc.contributor.authorCheung, Yam-fung, Hilaire-
dc.contributor.author張任丰-
dc.date.accessioned2019-03-01T03:44:45Z-
dc.date.available2019-03-01T03:44:45Z-
dc.date.issued2018-
dc.identifier.citationCheung, Y. H. [張任丰]. (2018). Immunocapture laser ionization mass spectrometry and liquid chromatography mass spectrometry for protein marker analysis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/267761-
dc.description.abstractMonitoring of protein marker levels is invaluable in clinical applications, such as disease diagnosis and drug development. Matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS) is widely employed in protein marker analysis, but it suffers from poor sensitivity and limits of detection for high mass protein analysis. Here, an immunocapture laser ionization mass spectrometry (LI-MS) assay using gold nanoparticles (AuNPs) conjugated antibodies for indirect protein detection was developed. With three characteristic properties, including localized surface plasmon resonance, high atom density and facile surface chemistry, a AuNP as an ion reservoir can generate abundant Au cluster ions for signal amplification upon laser ionization, and conjugate with antibody for targeting specific proteins. In this study, human chorionic gonadotropin (hCG) was used for method development. Factors that affect antibody density on an antibody array and background signal, including silanization time, antibody printing condition, and blocking agent were investigated. Based on the experimental conditions optimized in the current study, the limit of detection for hCG was determined to be 0.38 fmol. Moreover, the study revealed that increasing the size of AuNPs can improve sensitivity, with 80 nm AuNPs having 14 times higher sensitivity than 20 nm AuNPs. The developed immunocapture LI-MS assay was then employed to detect PML-RARA fusion protein in NB4 cells, a 120 kDa biomarker for acute promyelocytic leukemia (APL) diagnosis, where its high mass makes it difficult to be detected using MALDI-MS. In the study, the limit of detection for PML-RARA was found to be 10,000 NB4 cells, outperforming conventional Western blot analysis. Furthermore, the feasibility of silver nanoparticle (AgNP) as an ion reservoir for signal amplification was explored, with three times higher sensitivity compared with AuNPs of equal particle size. The results demonstrated the capability of the immunocapture LI-MS assay in trace protein detection in biological samples with complex matrices. Finally, MS-based proteomics with tandem mass tags labelling was adopted to investigate the effect of arsenic trioxide (ATO) on protein expression levels in NB4 cells. In the study, 2,226 proteins were identified across three biological replicates. Pathway enrichment analysis conducted on 170 significantly upregulated proteins and 106 downregulated proteins revealed that spliceosome, carbon metabolism, and mRNA surveillance pathway and decay were upregulated, while separation of sister chromatids and protein processing in endoplasmic reticulum were downregulated upon ATO treatment. Furthermore, the study observed a two-fold decrease in PML level after ATO treatment, in accordance with the immunocapture LI-MS assay results. Therefore, MS-based proteomics combined with immunocapture LI-MS assay can constitute an attractive tool for cross validation in proteomic analysis.-
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.lcshHigh performance liquid chromatography-
dc.subject.lcshMass spectrometry-
dc.subject.lcshSpectra - Proteins-
dc.titleImmunocapture laser ionization mass spectrometry and liquid chromatography mass spectrometry for protein marker analysis-
dc.typePG_Thesis-
dc.description.thesisnameMaster of Philosophy-
dc.description.thesislevelMaster-
dc.description.thesisdisciplineChemistry-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2019-
dc.identifier.mmsid991044081524203414-

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