<p> Biological thiols are a class of labile and redox-active metabolite with
significant interest to biomedical research due to their involvement in redox mechanisms
of cell signaling and physiological control. As a result of oxidative stress, levels of
various reduced thiols and oxidized disulfides are altered, which disrupts major cellular
regulation pathways modulating protein function and gene expression. Thus, analysis of
thiols in biological fluids is essential for understanding the role of oxidative stress and
thiol dysregulation in aging and human diseases. However, reliable ex-vivo thiol
determination is challenging due to their low abundance and susceptibility to auto-oxidation
and thiol-disulfide exchange reactions. In this thesis, capillary electrophoresis-electrospray
ionization-mass spectrometry (CE-ESI-MS) in conjunction with maleimide
labeling is developed as an integrative strategy for comprehensive plasma thiol redox
status analysis for metabolomics. Maleimide labeling helps to address both major
constraints in thiol analysis by stabilizing free sulfhydryl groups as their thioether adducts
while improving ionization efficiency and analytical sensitivity. This enhancement in
ionization efficiency can be quantitatively predicted based on relative changes in
fundamental physicochemical properties of thiols that occur upon covalent derivatization
when using multivariate calibration. On-line sample preconcentration together with
thiol-selective labeling using a cationic quaternary ammonium maleimide analog allowed
for simultaneous analysis of reduced thiols and intact oxidized disulfides by CE-ESI-MS
with low nanomolar detection limits of 8-30 nM. Improved identification of unknown
low abundance thiols and other classes of polar metabolites is also demonstrated by prediction of relative migration times in CE that is complementary to ESI-MS.
Comprehensive plasma thiol speciation together with untargeted profiling of polar
metabolites provides a novel platform for holistic understanding of complex changes in
metabolic networks associated with thiol dysregulation and/or nutritional intervention for
the prevention or treatment of human disorders. </p> / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/19189 |
| Date | 09 1900 |
| Creators | D' Agostino, Lisa |
| Contributors | Britz-McKibbin, Philip, Chemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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