Desorption electrospray ionization (DESI) is an ambient ionization technique for mass spectrometry. This solvent based desorption ion source has wide applicability in surface analysis with minimal sample preparation. Interest in improving detection limits, broadening applications, and increasing the spatial resolution for chemical imaging has led to studies of the DESI mechanism. An inverted microscope has been used to image interactions between the DESI spray and test analytes on a glass surface. Microscopic images recorded with millisecond time resolution have provided important insights into the processes governing analyte transport and desorption. These insights are the basis of a rivulet-based model for desorption that differs significantly from the widely-accepted momentum transfer model.

Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.

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This dissertation details an investigation into the use of laser pulses from a titanium:sapphire regenerative amplifier laser system to vaporize analytes in ambient air for mass spectral analysis. The laser system was modified to operate in one of two distinct modes. In femtosecond (fs) mode the laser produced 2.5 mJ, ~60 fs laser pulses centered at 800 nm. In nanosecond (ns) mode the laser produced 2.4 mJ, ~10 ns laser pulses centered at 800 nm. Using appropriate optical components the laser pulse energy was attenuated to achieve pulses varying from 0.15 mJ to 2.0 mJ. Laser pulses were used to vaporize liquid and solid samples on different substrates. The laser vaporized material was captured and ionized by an electrospray source and then detected via a mass spectrometer instrument. It was discovered that samples on glass substrate could be vaporized by fs laser pulses, but not by ns laser pulses. Samples on metal substrate were successfully vaporized by both fs and ns laser pulses. Low energy ns laser pulses were less efficient than fs laser pulses of the same energy for vaporizing off metal substrate. A comparison of vaporization from aluminum, copper and stainless steel substrates revealed limited vaporization from copper by ns laser pulses. The electrospray ionization (ESI) mass spectral response of wet and dry proteins on stainless steel was similar for both fs and ns laser pulses. Experiments to test the capabilities of ns laser electrospray mass spectrometry (ns-LEMS) revealed that sample vaporization was limited to analysis on metal surfaces. This dissertation details methods for femtosecond laser electrospray ionization (fs-LEMS) to be used to quantify non-covalent protein-ligand interactions. Hen egg white lysozyme (HEWL) and N,N’,N”-triacetylchitotriose (NAG3) interactions were quantified via dissociation constant (Kd) measurements. The Kd for HEWL and N,N’,N”,N”’-tetraacetylchitotetraose (NAG4) were also measured. This dissertation also reports a miniaturized flowing atmospheric pressure afterglow (micro-FAPA) for use as an alternative ionization source of fs-laser vaporized analytes. Loratadine pills were vaporized and reacted with the gas stream from the micro-FAPA source to generate ions which were then detected by a mass analyzer. The ions detected varied in distribution as a response to the distance the sample was vaporized from the ion source. Complexed samples were tested and molecular assignments were difficult due to the numerous pathways for ion formation. The use of an ion filter to decrease the energy imparted on sample molecules during the ionization process of the micro-FAPA is also reported. / Chemistry

This dissertation elucidates a greater understanding of protein folding and unfolding processes during the lifetimes of electrospray and nano-spray droplets in laser electrospray mass spectrometry (LEMS) and nano-laser electrospray mass spectrometry (nano-LEMS) measurements, respectively. The similarity in mass spectral features obtained from conventional electrospray measurements for supercharged proteins with those of LEMS measurements suggested that supercharging phenomena occurs in the electrospray droplets during the droplet desolvation process. It was observed that the laser vaporization of protein from condensed phase into the electrospray droplets containing denaturing electrospray solution and a supercharging reagent resulted in the increase in ion abundance of higher charge states in comparison with electrospray measurements. Conversely, the addition of solution additives with varying gas phase basicity in the electrospray solvent resulted in charge reduction for unfolded protein upon laser vaporization from condensed phase into the charged electrospray droplets. The extent of charge reduction and the fraction of folded protein within the electrospray droplets was found to be dependent upon both the extent of protein denaturation in the solution prior to laser vaporization and the gas phase basicity of solution additives. The ability of the LEMS technique to analyze molecules from solution with high matrix effects was established by the successful detection of protein molecules from solution with high salt concentration. Experiments with LEMS enabled the detection of a protonated protein feature as the dominating peak in the mass spectra for up to 250 mM sodium chloride while conventional electrospray resulted in predominantly salt-adducted features, with suppression of the protonated protein ions for the salt concentration of 5 mM. This dissertation also expanded upon the use of a reaction system to measure the lifetimes of laser vaporized liquid droplets coupled with electrospray and nano-spray postionization mass spectrometry. Electrospray and nanospray droplet lifetimes were measured to be 4.5±0.6 ms and 1.4±0.3 ms using LEMS and nano-LEMS measurements, respectively. Time dependent protein folding measurements using LEMS revealed intermediate states during protein folding processes which are often limited in conventional electrospray measurements where bulk solution in manipulated (change in pH) to achieve protein folding. / Chemistry

This dissertation elucidates the instrumentation and application of a hybrid ambient ionization source, laser electrospray mass spectrometry (LEMS), for the direct analysis and molecular imaging of biological tissue without matrix deposition. In LEMS, laser pulses from a Ti:Sapphire laser amplifier (60 fs, 800 nm, and 1 mJ) interact with surface analytes and transfer them from the condensed phase into the gas phase without the requirement of either exogenous matrix or endogenous water in the sample. The laser vaporized analytes are captured and ionized by an electrospray source, and finally detected by a mass analyzer. It was found that a turn-key, robust femtosecond fiber laser with longer wavelength, longer duration, and lower pulse energy at 1042 nm, 425 fs, and 50 µJ, respectively, provided comparable results with the Ti:Sapphire laser. Vaporization of intact, dried or aqueous cytochrome c and lysozyme samples was demonstrated by the fiber laser. A charge states distribution at lower charge states indicating folded conformation of proteins and the hemoglobin α subunit-heme complex from whole blood was observed. Endogenous anthocyanins, sugars, and other metabolites were detected and revealed the anticipated metabolite profile for the flower petal and leaf samples by the fiber laser. Phospholipids, especially phosphatidylcholine, were identified from a fresh mouse brain section sample. These lipid features were suppressed in both the fiber laser and Ti:Sapphire LEMS measurement in the presence of optimal cutting temperature compounds which are commonly used in animal tissue cryosectioning. This dissertation also details the design of an automated mass spectrometry imaging source based on the Ti:Sapphire LEMS. The laser, translation stage, and mass analyzer are synchronized and controlled using a customized user interface to enable step-by-step scanning of the area of interest on a given tissue sample. The imaging source is coupled with a high resolution accurate mass quadrupole time-of-flight (QTOF) mass analyzer with tandem mass analysis capability. A lateral resolution of 60 µm was demonstrated on a patterned ink film by LEMS imaging. Plant metabolites including sugar and anthocyanins were directly imaged from a leaf sample. Small metabolites, lipids and proteins were simultaneously imaged from a single tissue section of a pig liver sample. Biomarkers of blood-brain barrier damage and traumatic brain injury (TBI) that occurred during the injury were detected and imaged from a TBI mouse brain. The loading values from principal component analysis (PCA) were shown to be useful for identification of features of interest from the large LEMS imaging dataset. / Chemistry

Over the last few decades, membrane introduction mass spectrometry (MIMS) has been established as a robust tool for the on-line continuous monitoring of trace gases and volatile organic compounds. However, the range of amenable anlaytes has been limited by the need for molecules to pervaporate into a gaseous acceptor phase, or high vacuum environment of a mass spectrometer. This thesis expands the range of amenable analytes for MIMS to include larger, less volatile molecules (e.g., 200 to 500 Da), such as pharmaceuticals, persistent organic pollutants, and small biomolecules. This was achieved through the use of a liquid|membrane|liquid interface. We distinguish the technique from conventional MIMS, which uses a gaseous acceptor phase, by inserting the prefix ‘condensed phase’ to emphasize the use of a solvent acceptor phase – thus yielding CP-MIMS. An initial flow-cell interface with a methanol acceptor phase was characterized, yielding detection limits for model analytes in pptr to ppb, and analyte response times from 1-10 minutes. The flow cell interface was miniaturized into an immersion style CP-MIMS probe (~2 cm), which allowed for analysis of smaller volume samples and improved membrane washing capabilities. Comparable detection limits were observed for the immersion probe, however, it was noticed that significant analyte depletion was observed for samples under 2 mL. In addition, each of the developed membrane interfaces were observed to suffer from ionization suppression effects from complex samples when paired with ESI. Several strategies for mitigating ionization suppression using CP-MIMS are presented, including the use of a continuously infused internal standard present within the acceptor solvent. The developed CP-MIMS system was challenged with the analysis of naphthenic acids (a complex mixture of aliphatic carboxylic acids) directly in contaminated real-world samples. The method used negative ESI to rapidly screen and mass profile aqueous samples for naphthenic acids (as [M-H]-), with sample duty cycles ~20 min. However, it was found that Negative ESI did not differentiate hydroxylated and carboxylated analytes, and both species contributed signal to the total naphthenic acid concentration. To increase method specificity for carboxylic acids, barium ion chemistry was used in conjunction with positive ion tandem mass spectrometry. Common product ions were used to quantify carboxylated analytes, while a qualifier ion was used to confirm the functionality. The increased selectivity afforded by the barium ion chemistry was at the cost of a modest increase in detection limits. CP-MIMS has been established as a technique capable of the direct analysis of real-world samples, and shows promise as a rapid screening method for amenable environmental contaminants and/or biomolecules. / Graduate / 0486 / 0485 / kyle.duncan@viu.ca

Information about protein structures is important in many areas of life sciences, including structure-based drug design. Gas phase methods, like electrospray ionization and mass spectrometry are powerful tools for the analysis of molecular interactions and conformational changes which complement existing solution phase methods. Novel techniques such as single particle imaging with X-ray free electron lasers are emerging as well. A requirement for using gas phase methods is that we understand what happens to proteins when injected into vacuum, and what is the relationship between the vacuum structure and the solution structure. Molecular dynamics simulations in combination with experiments show that protein structures in the gas phase can be similar to solution structures, and that hydrogen bonding networks and secondary structure elements can be retained. Structural changes near the surface of the protein happen quickly (ns-µs) during transition from solution into vacuum. The native solution structure results in a reasonably well defined gas phase structure, which has high structural similarity to the solution structure. Native charge locations are in some cases also preserved, and structural changes, due to point mutations in solution, can also be observed in vacuo. Proteins do not refold in vacuo: when a denatured protein is injected into vacuum, the resulting gas phase structure is different from the native structure. Native structures can be protected in the gas phase by adjusting electrospray conditions to avoid complete evaporation of water. A water layer with a thickness of less than two water molecules seems enough to preserve native conditions. The results presented in this thesis give confidence in the continued use of gas phase methods for analysis of charge locations, conformational changes and non-covalent interactions, and provide a means to relate gas phase structures and solution structures.

<p>Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been established as a powerful tool for qualitative and quantitative analysis of biomolecules. However, mass spectrometric analysis of biomolecules is often limited by poor ionization efficiency of analyte for sensitive detection and limited fragmentation for structural characterization. Over the years, various solution phase as well as gas-phase derivatization techniques, have been coupled with MS to increase the ionization efficiency and facilitate the formation of structural informative fragment ions. The research presented in this dissertation falls into two major parts; focusing on method development and application of radical chemistry for enhanced biomolecule analysis on an ESI-MS/MS platform. In the first part, a method of rapid charge tagging of neutral lipids (e.g. sterols, glycerides) with a thiol radical-based charge tag is developed, followed by comprehensive analysis via ESI-MS/MS without the use of a chromatographic separation (shotgun lipidomics). This charge tagging is performed in an easily constructible fused silica capillary-based microflow photo-reactor which is relatively low in cost and requires no instrument modifications. This method significantly enhances the ionization efficiency of the neutral lipids for sensitive MS detection (pM range). This method can be applied to the small volume of biological complex samples (e.g. 1 µL plasma) and doesn’t require extensive sample pretreatment procedure (analysis time of 2 min vs. traditional >60 min on GC-MS and HPLC-MS systems). Furthermore, the derivatized neutral lipids can also be fragmented via soft collision-induced dissociation to obtain fatty acyl chain composition of the neutral lipids (sterol esters, diacylglycerols, triacylglycerols, etc.) for structural characterization. This can especially be useful for determination for fatty acyl compositional isomers in neutral lipids for analysis related to biomarker detection. The characteristic fragmentation pattern of tagged neutral lipids has also been utilized for quantitation of lipids from biological mixture samples. Initial application of this method has shown alteration in the concentration of diacylglycerol lipid species in clinical samples of Type 2 Diabetes Mellitus patients, suggesting the potential of understanding the biological roles of such lipids in insulin resistance. </p> <p>In the second part, a unique approach of radical-induced disulfide bond cleavage in peptides and proteins is demonstrated. Using 254 nm UV emission, acetone was used as a photoinitiator to initiate secondary radical formation i.e. hydroxyalkyl radical, from alcohol co-solvents used for electrospray. These radicals can then be used to efficiently cleave the disulfide bonds (R-S-S-R) in peptide/proteins to give reduced reaction products (RSH) at the cleavage site. Upon soft collision-induced dissociation, the reduced product gave abundant <i>b-</i> and <i>y-</i> type fragment ions for complete or enhanced sequence coverage as compared to intact disulfide-linked peptides and proteins. With the use of a simple microflow photo-reactor, this radical based approach can also be coupled with infusion ESI-MS/MS for a rapid online-based peptide and protein analysis. The yield for disulfide bond reduction was almost 100% within less than 5 s of UV irradiation. Furthermore, by adjusting the UV irradiance time, different degrees of partial reduction could be achieved, which greatly facilitated the disulfide linkage mapping in peptides and proteins with multiple disulfide bonds. This method has been incorporated with both bottom-up and top-down approach for protein analysis for unraveling the molecular complexity, quantifying and deep sequencing of disulfide-linked proteins.</p>

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