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This thesis investigates the exposure of humans to organic combustion products. Combustion of natural and anthropogenic materials can lead to highly complex mixtures of gas-phase and particle-bound chemical compounds, whose composition and health effects have been studied extensively. Nevertheless, the analysis of other potentially toxic products remains a challenge due to lack of analytical standards and methodologies. The research that encompasses this thesis is a progression from the analysis of known combustion products to the identification of previously unknown products. Targeted analytical techniques, such as gas chromatography tandem mass spectrometry (GC-MS/MS), were utilized to evaluate firefighter exposure to wood smoke chemicals during training exercises. The results suggest that a subset of the firefighters were at higher risk of exposure which could be related to specific operational roles and the use of personal protective gear. Comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCGC-TOF) was used for the identification of novel wood smoke markers and the results indicate that firefighters are equally exposed to gas-phase and particle phase compounds.. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry is a non-targeted technique that is complementary to GCGC. Together, these tools enabled the identification of a suite of halogenated PAHs (haloPAHs) in samples obtained from the Plastimet Inc. fire, one the largest industrial fires in North America. HaloPAHs are similar in structure to toxic polychlorinated dibenzo-p-dioxins (PCDDs), a notorious class iv of toxic chemicals, and they were detected at much higher concentrations. In addition, highly substituted and high molecular weight haloPAHs were detected for the first time in an environmental sample. Finally, negative ion atmospheric pressure chemical ionization (NI-APCI) was explored as an alternative ionization technique for the analysis of mixed bromo/chloro dioxins (PXDDs) in the ash sample. PXDDs, with 1550 possible congeners, are potentially more toxic than their chlorinated counterparts (PCDDs). NI-APCI derived structure diagnostic fragments enabled the differentiation of co-eluting PXDD isomers in the ash sample which has not been possible using traditional ionization techniques (EI/CI) associated with GC-MS. / Thesis / Doctor of Philosophy (PhD)

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In this work, tandem mass spectrometry (MS/MS)-based quantitative protocols were developed to facilitate differential protein expression analysis and biomarker discovery via a two-step sample interrogation strategy: (a) global protein profiling and differential expression analysis by spectral counting; and, (b) biomarker candidate validation by targeted screening, i.e., multiple reaction monitoring (MRM). Preliminary experiments were performed to evaluate the performance of the spectral counting method. The method proved to be applicable for proteins with spectral counts≥2, and a close-to-linear relationship between protein concentration and spectral count data was achievable at protein concentration levels <0.1 μM. The detection limit was 40-800 fmol. A protein/peptide library containing ~10,000 peptide entries that facilitates the development of future MRM experiments, was developed. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum, the peptide retention time, and the top 10 most intense product ions that correspond to a given parent peptide. Only proteins identified by at least two spectral counts are listed. An MRM experiment was performed to demonstrate the successful applicability of this peptide library for the identification of putative biomarkers in proteomic samples. / Master of Science

Call number: LD2668 .T4 1956 B87 / Master of Science

Investigating the structure of proteins and their interactions with other biomolecules or drug molecules, coupled with the consideration of conformational change upon binding, is essential to better understand their functions. Mass spectrometry (MS) is emerging as a powerful tool to study protein and peptide structure and interactions due to the high dynamic range, low sample consumption and high sensitivity of this technique, providing insight into the stoichiometry, intensity and stability of interactions. The hybrid technique of ion mobility-mass spectrometry (IM-MS) can provide insight into the conformations adopted by protein and peptide monomers and multimers, in addition to complexes resulting from interactions, which when coupled with molecular modelling can suggest candidate conformations for these in vacuo species and by inference their conformations in solution prior to ionisation and desolvation. The work presented in this thesis considers a number of different peptide and protein systems, highlighting how the combination of MS and IM-MS based techniques, in conjunction with other biophysical techniques such as circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM) and isothermal titration calorimetry (ITC) can provide insight into these dynamic systems. First a case study into the ability of MS and IM-MS to study disorder-to-order transitions is presented. The transcription factor c-MYC can only perform its function upon binding with its binding partner MAX; deregulation of c-MYC is, however, implicated in a number of human cancers. c-MYC and MAX comprise intrinsically disordered regions which form a leucine zipper upon binding. The work presented here focuses on the leucine zipper regions of both c-MYC and MAX, their individual conformations and changes upon binding. Inhibiting the c-MYC:MAX interaction is a current target for drug therapy and hence the inhibition of this interaction with a previously identified small drug-like molecule was also examined using these techniques, to determine if such an approach may be appropriate for investigation of future therapeutics. Next the ability of MS-based techniques to preserve, transmit and distinguish between multiple conformations of a metamorphic protein was examined. The chemokine lymphotactin has been shown to exist in two distinct conformations in equilibrium in a ligand-free state. The existence of such metamorphic proteins has called into question whether traditional structural elucidation tools have been inadvertently biased towards consideration of single conformations. Here, the potential of gas-phase techniques in the study of conformationally dynamic systems is examined through the study of wild type lymphotactin and a number of constructs designed either as a minimum model of fold or to mimic one of the distinct folds. Interactions between chemokines and glycosaminoglycans (GAGs) are thought to be essential for the in vivo activity of these proteins. The interactions between the distinctive chemokine lymphotactin and a model GAG were hence probed. As with the structural studies, additional protein constructs were considered either to represent the minimum model of fold, one distinct fold of the metamorphic protein or designed to diminish its GAG binding propensity. The ability of each construct to bind GAGs, the stoichiometry of the interactions and conformations adopted by the resulting complexes in addition to aggregation occurring upon the introduction of the GAG is considered. Finally, the similarities, with respect to structure and function, between the chemokine superfamily of proteins and the human β-defensin subfamily of antimicrobial peptides are considered. The tendency of human β-defensins 2 and 3 to bind a model GAG is examined; the stoichiometry of binding and conformations adopted and aggregation occurring here are considered and compared with that of chemokines.

Chemical imaging by mass spectrometry is a powerful approach by which to map spatial distributions of molecules to better understand their function in the system of interest. Over the last thirty years, MSI has evolved into a very powerful analytical tool for the investigation of chemically-complex samples including biological tissues, catalytic surfaces and thin layer chromatography plates, among many others. The work in this dissertation aimed to characterize existing MSI methods, while also developing novel instrumentation able to overcome the challenges found in a variety of applications. Different sample preparation and ionization techniques were evaluated to maximize detection of lipid species in brain tissues subjected to traumatic injury to better understand the biological processes involved. Next, differential mobility separation was coupled to an ambient MSI system that resulted in increased signal-to-noise ratios and image contrast. Third, bulky catalytic granite surfaces were imaged to determine specific mineral reactivity and demonstrate the ability of desorption electrospray ionization to image such samples. Fourth, a novel technique was developed names Robotic Plasma Probe Ionization (RoPPI), which uses a vision system-guided robotic arm to probe irregular surfaces for three dimensional surface imaging. Finally, a software program was developed to automatically screen MSI datasets acquired from thin layer chromatography separations for spot-like shapes corresponding to mixture components; this program was named DetectTLC. This research resulted in instrumentation advances for MSI that have enabled increased chemical diversity, enhanced sensitivity and image contrast, imaging of bulky or irregularly-shaped surfaces, and multivariate tools to facilitate data interpretation.