<p dir="ltr">Multiply-charged ions of intact biomolecules generated from electrospray ionization (ESI) have drawn researchers' interest in the field of native mass spectrometry (MS) for decades because these ions carry mass and charge information of the intact molecules and interactions among different units. However, the confinement of multiple charge states in a narrow range of <i>m/z</i> makes mass and charge assignments challenging, especially for analytes with a mass greater than 100 kDa. Gas-phase ion/ion reactions have proven to be powerful techniques that facilitate the interpretation of mass spectra of natively sprayed macromolecular analytes by manipulating the masses and charges of ions detected.</p><p dir="ltr">The proton-transfer reaction (PTR) is the most used gas-phase ion/ion reaction method. It utilizes perfluorinated PTR reagents to "grab" protons away from the analyte ions, thereby reducing their charges. A novel charge state manipulation technique called "ion parking," based on PTR, has been developed. In this method, ion signals are accumulated to one or a range of charge states by selectively inhibiting reactions between the target charge state and the PTR reagents via resonance excitation.</p><p dir="ltr">The multiply-charged ion attachment (MIA) reaction is another gas-phase ion/ion reaction approach. It utilizes the significant <i>m/z</i> displacement caused by the attachment of multiply-charged reagent ions, and it has been proven useful for mass analysis of heterogeneous macromolecular analytes with a mass greater than 1 MDa.</p><p dir="ltr">All gas-phase ion/ion reaction techniques require mutual storage of ions in opposite polarities within an electrodynamic quadrupole ion trap, such as a 3D quadrupole ion trap (QIT) or a linear quadrupole ion trap (LIT). Electrodynamic ion traps use high-voltage (HV) drive radio frequencies (RF) to trap ions in a quadrupolar field, typically employing a sinusoidal waveform (sine wave). A digital quadrupole ion trap (DIT) is an unconventional electrodynamic ion trap that uses a digital waveform (square wave) as the drive RF. The high agility of square waves makes DIT an ideal mass analyzer for studying high <i>m/z</i> ions resulting from gas-phase ion/ion reactions. This dissertation describes the development of a novel home-built digital dual-trap mass spectrometer and ion/ion chemistry studies of large biomolecules within the instrument.</p>

<p>Microglia are a population of specialized, tissue-resident immune cells that make up around 10% of total cells in our brain. They actively prune neuronal synapses, engulf cellular debris, and misfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta (Aβ) by the process of phagocytosis. During AD, microglia are unable to phagocytose Aβ, perhaps due to the several disease-associated changes affecting their normal function. Functional molecules such as lipids and metabolites also influence microglial behavior but have primarily remained uncharacterized to date. The overarching question of this work is, <i>How do microglia become dysfunctional in chronic inflammation</i>? To this end, we developed new chemical tools to better understand and investigate the microglial response to Aβ <i>in vitro</i> and <i>in vivo</i>. Specifically, we introduce three new tools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust method for expressing, purifying, and characterizing the protein. (2) A pH-sensitive fluorophore conjugate of Aβ (called Aβ^pH) was developed to identify and separate Aβ-specific phagocytic and non-phagocytic glial cells <i>ex vivo</i> and <i>in vivo</i>. (3) New lysosomal, mitochondrial, and nuclei-targeting pH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine, respectively) to visualize subcellular organelles in live microglia. Next, we asked, <i>What changes occur to the global lipid and metabolite profiles of microglia in the presence of Aβ in vitro and in vivo</i>? We screened 1500 lipids comprising 10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant changes in specific lipid classes with acute and prolonged Aβ exposure. We also identified a lipid-related protein that was differentially regulated due to Aβ <i>in vivo</i>. This new lipid reprogramming mechanism “turned on” in the presence of cellular stress was also present in microglia in the brains of the 5xFAD mouse model, suggesting a generic response to inflammation and toxicity. It is well known that activated microglia induce reactive astrocytes during inflammation. Therefore, we asked, <i>What changes in proteins, lipids, and metabolites occur in astrocytes due to their reactive state? </i>We provide a comprehensive characterization of reactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites. These microglia and astrocytes datasets will be available to the scientific community as a web application. We propose a final model wherein the molecules secreted by reactive astrocytes may also induce lipid-related changes to the microglial cell state in inflammation. In conclusion, this thesis highlights chemical neuroimmunology as the new frontier of neuroscience propelled by the development of new chemical tools and techniques to characterize glial cell states and function in neurodegeneration.</p>

<div>Vibrational spectroscopic imaging has become an emerging platform for chemical visualization of biomolecules and materials in complex systems. For over a century, both Raman and infrared spectroscopy have demonstrated the capability to recognize molecules of interest by harnessing the characteristic features from molecular fingerprints. With the recent development of hyperspectral vibrational spectroscopy imaging, which records the chemical information without sacrificing the spatial-temporal resolution, numerous discoveries has been achieved in the field of molecular and cellular biology. Despite the ability to provide complimentary chemical information to Raman-based approaches, infrared spectroscopy has not been extensively applied in routine studies due to several fundamental limitations: 1). the poor spatial resolution; 2). inevitable strong water absorption; 3). lack of depth resolution.</div><div>Mid-infrared photothermal (MIP) microscopy overcame all the above mentioned problems and for the first time, enabled depth-resolved in vivo infrared imaging of live cells, microorganisms with submicrometer spatial resolution. The development of epi-detected MIP microscopy further extends its application in pharmaceutical and materials sciences. With the deployment of difference frequency generation and other nonlinear optical techniques, the spectral coverage of the MIP microscopy was significantly enhanced to enable chemical differentiation in complex systems across the broad mid-infrared region. In addition to the efforts to directly improve the performance of MIP microscopy, a novel quantitative phase imaging approach based on polarization wavefront shaping via custom-designed micro-retarder arrays was developed to take advantage of the highly sensitive phase measurement in combination with the photothermal effect. Besides, the extended depth-of-field and multifocus imaging enabled by polarization wavefront shaping could both improve the performance of MIP microscopy for volumetric imaging.</div>

In the area of forensic chemistry, improved or new analysis methods are continually being investigated. One common and powerful technique used in forensic chemistry is wall-coated open-tubular column (WCOT) gas chromatography with electron ionization single quadrupole mass spectrometry (GC-MS). Improvements to and effectiveness of alternatives to this instrumental platform were explored in an array of parallel inquiries. The areas studied included the column for the chromatographic separation, the universal detection method employed, and the fragmentation method used to enhance molecular identification. <br><br>Superfine-micropacked capillary (SFµPC) columns may provide an alternative to commercial packed GC columns and WCOT GC columns that combines the benefits of the larger sample capacity of packed columns and the benefits of the excellent separation capabilities and mass spectrometry (MS) flow rate compatibility of WCOT columns. SFµPC columns suffer from high inlet pressure requirements and prior reported work has required specialized instrumentation for their use. Fabrication of and chromatography with SFµPC GC columns was successfully achieved with typical GC-MS instrumentation and within the flow rate limit of a MS. Additionally, the use of higher viscosity carrier gasses was demonstrated to reduce the required inlet pressure for SFµPC GC columns.<br><br>Recently, a new vacuum ultraviolet spectrometer (VUV) universal detector has been commercialized for GC. The ability of VUV detectors to acquire absorbance spectra from 125 nm to 430 nm poses a potential alternative to MS. As such, GC-VUV provides an exciting potential alternative approach to achieving excellent quantitative and qualitative analysis across a wide range of analytes. The performance of VUV and MS detectors for forensic analysis in terms of quantitative and qualitative analysis was compared. Analysis of alkylbenzenes in ignitable liquids was explored, which can be important evidence from suspected arson fires and are difficult to differentiate with MS. The VUV detector was found to have superior specificity and comparable sensitivity to the MS detector in scan mode.<br><br>Addition of thermolysis (Th) as an orthogonal fragmentation pathway provides the opportunity to increase the differences between MS fragmentation patterns. Fragmentation has been widely established to aid in identification of molecules with MS by providing characteristic fragments at characteristic relative abundances. However, molecules with very similar structures do not result in sizable spectral differences in all cases with typical MS fragmentation techniques. A series of Th units were fabricated and integrated into GC-Th-MS instruments. Th-MS was conducted with the thermally labile nitrate esters across a range of instrumentation and thermal conditions.<br>

<p>The rapid analysis of complex samples using mass spectrometry (MS) provides valuable information in both point-of-care (e.g. drug testing) and laboratory-based applications, including the generation of spectral libraries for classification of biosamples, the identification of biomarkers through large-scale studies, as well as the synthesis and bioactivity assessments of large compound sets necessary for drug discovery. In all these cases, the inherent speed of MS is attractive, but rarely fully utilized due to the widespread use of sample purification techniques prior to analysis. Ambient ionization methodologies can help circumvent this drawback by facilitating high-throughput qualitative and quantitative analysis directly from the complex samples without any need for work-up. For instance, the use of swabs or paper substrates allows for rapid identification, quantification, and confirmation, of drugs of abuse from biofluids or surfaces of forensic interest in a matter of minutes, as described in the first two chapters of this dissertation. Faster analysis can be achieved using an automated desorption electrospray ionization (DESI) platform which allows for the rapid and direct screening of complex-sample microarrays with throughputs better than 1 sample per second, giving access to rich spectral information from tens of thousands of samples per day. The development of the bioanalytical capabilities of this platform, particularly within the context of drug discovery (e.g. bioactivity assays, biosample analysis), is described across most other chapters of this dissertation. The use of DESI, a contactless ambient ionization method developed in our laboratory and whose 20 years of history are overviewed in the introduction of this document, provides an additional advantage as the secondary microdroplets generated through the DESI process act as reaction vessels that can accelerate organic reactions by up to six orders of magnitude, facilitating on-the-fly synthesis of new compounds from arrays of starting materials. Unique implications of this microdroplet chemistry in the prebiotic synthesis of peptides and spontaneous redox chemistry at air-solution interfaces, together with its practical applications to the synthesis of new drug molecules, are also overviewed. The success obtained with the first automated DESI-MS system, developed within the DARPA Make It program, led to increased interest in a new-generation platform which was designed over the past year, as overviewed in the last section of this dissertation, and which is currently being installed for validation prior to the transfer of the technology to NCATS, where we anticipate it will make a significant impact through the consolidation and acceleration of the early drug discovery workflow.</p>

<p>Canines can detect different diseases simply by smelling different biological sample types, including  urine,  breath  and  sweat.  This  has  led  researchers  to  try  and  discovery  unique  volatile  organic compound (VOC) biomarkers. The power of VOC biomarkers lies in the fact that one day they may be able to be utilized for noninvasive, rapid and accurate diagnostics at a point of care using  miniaturized  biosensors.  However,  the  identity  of  the  specific  VOC  biomarkers  must  be  demonstrated before designing and fabricating sensing systems. Through  an  extensive  series  of  experiments,  VOCs  in  urine  are  profiled  by  solid  phase  microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) to identify biomarkers for breast cancer using murine models. The results from these experiments indicated that  unique  classes  of  urinary  VOCs,  primarily  terpene/terpenoids  and  carbonyls,  are  potential  biomarkers  of  breast  cancer.  Through  implementing  chemometric  approaches,  unique  panels  of  VOCs  were  identified  for  breast  cancer  detection,  identifying  tumor  location,  determining  the  efficacy of dopaminergic antitumor treatments, and tracking cancer progression. Other diseases, including COVID-19 and hypoglycemia (low blood sugar) were also probed to identify volatile biomarkers present in breath samples.  VOC biomarker identification is an important step toward developing portable gas sensors, but  another  hurdle  that  exists  is  that  current  sensors  lack  selectivity  toward  specific  VOCs  of  interest.  Furthermore,  testing  sensors  for  sensitivity  and  selectivity  is  an  extensive  process  as  VOCs  must  be  tested  individually  because  the  sensors  do  not  have  modes  of  chromatographic  separation or compound identification. Another set of experiments is presented to demonstrate that SPME  fibers  can  be  coated  with  materials,  used  to  extract  standard  solutions  of  VOCs,  and  analyzed  by  GC-MS  to  determine  the  performance  of  various  gas  sensing  layers.  In  the  first  of  these  experiments,  polyetherimide  (PEI)  was  coated  onto  a  SPME  fiber  and  compared  to  commercial polyacrylate (PAA) fibers. The second experiment tuned the extraction efficiency of polyvinylidene fluoride (PVDF) - carbon black (CB) composites and showed that they had higher sensitivity  for  urinary  VOC  extraction  relative  to  a  polydimethylsiloxane  (PDMS)  SPME  fiber.  These results demonstrate SPME GC-MS can rapidly characterize and tune the VOC adsorption capabilities of gas sensing layers. </p>

<p dir="ltr">The beautiful complexity of our world is manifested in how macro- and even planetary-scale processes are essentially completely determined and regulated by chemical and physical transformations happening at the micro- and nanoscale. The introduction and subsequent development of optical microscopy methods have provided us with a unique opportunity to visualize, probe, and sometimes even control these processes that are too small to be seen by the human eye by their nature.</p><p dir="ltr">Among the great variety of truly impressive advances in microscopy instrumentation, two techniques stand out in their widespread and usefulness. First of them, fluorescence imaging has completely revolutionized the study of biological specimens and living systems due to its unprecedented single-molecule sensitivity and resolution combined with video-rate imaging capability. On the other hand, chemical imaging in the mid-infrared region provides an unmatched amount of chemical information enabling label-free mapping of the spatial distribution of various classes of biological molecules. However, each of these techniques falls short where the other excels. For example, despite its high resolution and sensitivity, fluorescence imaging does not carry direct chemical information and relies on labeling specificity, while infrared microscopy is diffraction-limited at the resolution of several micrometers and suffers from low penetration depth in aqueous solutions.</p><p dir="ltr">This dissertation introduces a novel imaging method designed to combine the advantages of fluorescence imaging and infrared spectroscopy. Fluorescence-detected photothermal mid-IR (F-PTIR) microscopy is presented in <b>chapter 1</b> as a technique enabling sub-diffraction chemically-specific microscopy by detecting local temperature-induced fluctuations in fluorescence intensity to inform on localized mid-infrared absorption. F-PTIR applications in targeted biological microspectroscopy (<b>chapter 1</b>) and pharmaceutical materials (<b>chapters 2 and 3</b>) analysis are demonstrated to highlight the potential of this new method. Furthermore, instrumentation developments relying on modern radiation sources such as dual-comb quantum cascade laser and synchrotron infrared radiation are shown to improve spectral acquisition speed (<b>chapter 4</b>) and spectral coverage (<b>chapter 5</b>), respectively, to extend the application range of F-PTIR.</p>

<p dir="ltr">Mass spectrometry imaging (MSI) is a powerful technique for visualizing the distribution of molecules within biological samples. Advancements in MSI instrumentation and computational tools have enabled the impactful applications of this technique across various fields including clinical research, drug discovery, forensics, microbiology, and natural products. Nanospray desorption electrospray ionization (nano-DESI), an ambient localized liquid extraction ionization technique, has proven valuable to the MSI community. Nano-DESI has been used for imaging of various molecules in biological samples including drugs, metabolites, lipids, N-linked glycans, and proteins.</p><p dir="ltr">My research has been focused on expanding the sensitivity and specificity of nano-DESI for biomolecular imaging. One of the newly developed methods employs ammonium fluoride NH₄F as a solvent additive to enhance the sensitivity of nano-DESI for the analysis of lipids in negative ionization mode. Secondly, methods were developed for the spatial mapping of isobaric and isomeric species in biological tissues by implementing nano-DESI MSI on a triple quadrupole (QqQ) mass spectrometer. This work used multiple reaction monitoring (MRM) mode of a QqQ with unit mass resolution to separate isobaric lipid species that require high mass resolving power and imaging of isomeric low-abundance species in tissue sections. Next, I demonstrate nano-DESI as a liquid extraction technique for imaging of N-linked glycans within biological tissue sections. Lastly, the spatial distribution of eicosanoids and specialized pro-resolving mediators (SPMs) in a mouse model for acetaminophen-induced liver injury (AILI) provides insights into the inflammation and resolution phases of AILI. Collectively, these developments have advanced the sensitivity, chemical specificity, and molecular coverage of nano-DESI for imaging of different classes of molecules in biological tissues.</p>

<p dir="ltr">Atmospheric organic aerosols (OA) have diverse compositions and undergo complex reactions and transformations within the atmosphere, leading to profound impacts on air quality, climate, and atmospheric chemistry. In particular, these aerosols play an important role in Earth's effective radiative forcing (ERF) through interactions with solar radiation, absorbing and scattering sunlight and terrestrial radiation. These interactions result in a warming and cooling effect on the climate, respectively. This dissertation seeks to unravel the intricate molecular characteristics of atmospheric OA, focusing specifically on its light-absorbing components, known as ‘Brown Carbon’ (BrC), and aims to comprehend its dynamic interplay within the atmosphere. The research employs state-of-the-art multi-modal mass spectrometry techniques to investigate atmospheric OA derived from the combustion of fossil fuels and biomass burning. Through a combination of controlled laboratory experiments and real-world sample analyses, these works provide molecular-level insights crucial for source apportionment and predictive modeling of OA fate. Chapter 2 details the instrumentation and data analysis methods, laying a robust foundation for subsequent chapters.</p><p dir="ltr">Chapter 3 delves into the investigation of smoldering-phase biomass burning organic aerosols (BBOA) and introduces an innovative fractionation method for high-level molecular characterization, targeted to streamline source apportionment of BBOA. This chapter also presents an extensive assessment of particle-to-gas partitioning of BBOA, providing valuable information for modeling atmospheric lifetimes and fate. In Chapter 4, a comparative analysis of BBOA from wild and agricultural fires is conducted, employing advanced molecular characterization techniques. Chapter 5 showcases the synergistic use of multi-modal mass spectrometry techniques to probe the chemical evolution of individual BBOA components. Finally, Chapter 6 examines the molecular analysis of secondary OA (SOA) generated from the photooxidation of a fossil-fuel proxy.</p><p dir="ltr">The comprehensive molecular-level studies presented contribute essential insights for climate modeling, aiding in resolving uncertainties associated with OA's impact on global ERF. The research not only challenges existing analytical methods but also introduces novel approaches for obtaining relevant information about atmospheric OA components. Overall, this work advances our understanding of the intricate dynamics of atmospheric aerosols, facilitating more accurate climate predictions and addressing uncertainties surrounding their net radiative impact.</p>

In forensic chemistry, a quicker and more accurate analysis of a sample is always being pursued. Speedy analyses allow the analyst to provide quick turn-around times and potentially decrease back-logs that are known to be a problem in the field. Accurate analyses are paramount with the futures and lives of the accused potentially on the line. One of the most common methods of analysis in forensic chemistry laboratories is gas chromatography, chosen for the relative speed and efficiency afforded by this method. Two major routes were attempted to further improve on gas chromatography applications in forensic chemistry.<br> The first route was to decrease separation times for analysis of ignitable liquid residues by using micro-bore wall coated open-tubular columns. Micro-bore columns are much shorter and have higher separation efficiencies than the standard columns used in forensic chemistry, allowing for faster analysis times while maintaining the expected peak separation. Typical separation times for fire debris samples are between thirty minutes and one hour, the micro-bore columns were able to achieve equivalent performance in three minutes. The reduction in analysis time was demonstrated by analysis of ignitable liquid residues from simulated fire debris exemplars.<br> The second route looked at a relatively new detector for gas chromatography known as a vacuum ultraviolet (VUV) spectrophotometer. The VUV detector uses traditional UV and far-ultraviolet light to probe the pi and sigma bonds of the gas phase analytes as well as Rydberg traditions to produce spectra that are nearly unique to a compound. Thus far, the only spectra that were not discernable were from enantiomers, otherwise even diastereomers have been differentiated. The specificity attained with the VUV detector has achieved differentiation of compounds that mass spectrometry, the most common detection method for chromatography in forensic chemistry labs, has difficulty distinguishing. This specificity has been demonstrated herein by analyzing various classes of drugs of abuse and applicability to “real world” samples has been demonstrated by analysis of de-identified seized samples.<br>