Bacteria cause significant morbidity and mortality throughout the world, including deadly diseases such as tuberculosis, meningitis, cholera, and pneumonia. Timely and accurate bacterial identification is critical in areas such as clinical diagnostics, environmental monitoring, food safety, water and air quality assessment, and identification of biological threat agents. At present, there is an established need for high throughput, sensitive, selective, and rapid methods for the detection of pathogenic bacteria, as existing methods, while nominally effective, have failed to sufficiently reduce the massive impact of bacterial contamination and infection. The work presented in this thesis focuses on addressing this need and augmenting conventional microorganism research through development of mass spectrometry (MS)-based proteomic applications. MS, a well established tool for addressing biological problems, offers a broad range of laboratory procedures that can be used for taxonomic classification and identification of microorganisms. These methods provide a powerful complement to many of the widely used molecular biology approaches and play critical functions in various fields of science. While implementation of modern biomolecule-identifying instrumentation, such as MS, has long been postulated to have a role in the microbiology laboratory, it has yet to be accepted on a large scale. Described in this document are MS methods that erect strong foundations on which new bacterial diagnostics may be based. A general introduction on key aspects of this work is presented in Chapter 1, where different approaches for detection of pathogenic bacteria are reviewed, and an overview regarding MS and microbial identification is provided. Chapter 2 presents the first implementation of microbial identification via rapid, open air Direct Analysis in Real Time MS (DART MS) to generate ions directly from microbial samples, including the disease-causing bacteria, Coxiella burnetii, Streptococcus pyogenes, and Escherichia coli. Chapter 3 expands on whole cell C. burnetii MS analysis and presents a rapid differentiation method to the strain-level for C. burnetii using mass profiling/fingerprinting matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and multivariate pattern recognition. Chapter 4 presents a unique "top-down" proteomics approach using 15N-labeled bacteriophage amplification coupled with MALDI-TOF MS as a detector for the rapid and selective identification of Staphylococcus aureus. Chapter 5 extends the idea of using isotopically labeled bacteriophage amplification by implementing a "bottom-up" proteomics approach that not only identifies S. aureus in a sample, but also quantifies the bacterial concentration in the sample using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) as a detector. In conclusion, Chapter 6, summarizes and contextualizes the work presented in this dissertation, and outlines how future research can build upon the experimentation detailed in this document.

The increasingly complex world of illicit chemistry has created a need for rapid, selective means of determining the threat posed by new drugs as they are encountered by law enforcement personnel. To streamline this process, the entirety of the problem, from the production of illicit drugs all the way to the final analysis have been investigated. A series of N-alkylated phenethylamine analogues were synthesized in a shotgun method and subjected to direct-infusion analysis. A range of products were detected without the need for time-consuming purification steps, which was extended to novel pharmacological and receptor-binding assays where mass spectrometry is used as a detector. This direct-infusion technique was also applied to studies of methamphetamine and fentanyl production to preemptively determine improvements to common reaction conditions and explore the origins of common impurities. The ability to utilize these rapid techniques directly from the fume hood has also been critically reviewed to highlight gaps in current research and opportunities for improvement. When combined, these studies seek to provide a means for rapid, simplified analysis of illicit drugs to improve the quality of data and dramatically increase throughput.

Despite the variety of successful reports of the preservation, recovery, and identification of archaeological proteins in general, there are few positive reports regarding mass spectrometry-based identification of ceramic-bound proteins. In large part, this shortage is due to the lack of consideration for the unique taphonomic histories of such residues and, in general, methods development. Further, because negative results are rarely published, there is no baseline to which results can be compared. This paper attempts to address these challenges via a multi-pronged approach that uses mass spectrometry and complementary approaches to evaluate ceramic-bound protein preservation in both controlled, actualistic experiments, and in archaeological artifacts. By comparing the results obtained from protein-spiked, experimentally-aged ceramic to those obtained from both faunal and ceramic archaeological materials, an enhanced perspective on protein preservation and subsequent recovery and identification is revealed. This perspective, focusing on taphonomy, reveals why negative results may be the norm for ceramic artifacts when non-targeted methods are employed, and provides insight into how further method development may improve the likelihood of obtaining positive results.

Presented in this thesis are two methods that are coupled to the instrumentation for the recovery and analysis of ultra-trace illicit drug residues. The electrostatic dust lifting process is coupled with nanomanipulation-nanospray ionization to retrieve drug particles off of hard surfaces for analysis. For the second method, drug residues from fingerprint impressions are extracted followed by analysis. The methodology of these hyphenated techniques toward forensic science applications is applied as to explore limits of detection, sensitivity, and selectivity of analytes as well as immediacy and efficiency of analysis. The application of nanomanipulation-coupled to nanospray ionization-mass spectrometry toward forensic science based applications is considered as future improvements to trace and ultra-trace analysis.

Ambient mass spectrometry (MS) is a new and growing sub-field in MS which has opened new research avenues, particularly for applications relating to the analysis of solid samples. Results on the implementation and application of ambient MS techniques including: desorption electrospray ionization (DESI) and direct analysis in real time (DART) indicated that these techniques could serve as complementary tools for the rapid qualitative screening of pharmaceuticals, allowing up to two orders of magnitude improvement in throughput compared to traditional methods such as liquid chromatography MS. The selectivity of DESI could be enhanced by performing the experiment in the reactive mode. In this mode, complexation reactions between reagents added to the spray solvent and analytes on the sample surface resulted in analyte stabilization, inhibiting fragmentation. They also resulted in a concomitant enhancement in the analyte surface activity, facilitating their evaporation from secondary droplets culminating in an improvement in sensitivity. Also for drug tablets analysis, the analyte signal dependency on DESI geometrical set-up variables could be mitigated following the careful and controlled addition of an isotopically labeled internal standard (IS) to the sample or by spraying samples with a pair of reagents with different affinities for the analyte. Either of these approaches resulted in an analyte-to-IS signal ratio (in the former) or an analyte complex ratio (in the later), which was largely independent of DESI experimental variables allowing quantitative analysis using this technique. DESI MS was also observed to be a very powerful tool for determining the 2-D distribution of various pharmaceutically important compounds on tablet and tissue surfaces. The ability to map the distribution of molecules of interest by DESI MS has very great implications in drug tablet quality control and in determining the role of chemical signals presented on tissue surfaces. DESI was observed to be limited to ionizing molecules of medium to high polarities without much limitation in terms of mass range, whereas DART was better suited for the analysis of molecules within a broader range of polarities, but within a more limited mass range (up to 800 Da approximately). These limitations were circumvented by implementing a novel multimode ambient ion source, desorption electrospray/metastable-induced ionization (DEMI), which combines various aspects of DESI and DART. Initial experiments with the DEMI ion source demonstrated its ability to enable the simultaneous analysis of molecules within a broader range of polarities and masses than DESI and DART alone.

The field of direct inject mass spectrometry includes a massive host of ambient ionization techniques that are especially useful for forensic analysts. Whether the sample is trace amounts of drugs or explosives or bulk amounts of synthetic drugs from a clandestine laboratory, the analysis of forensic evidence requires minimal sample preparation, evidence preservation, and high sensitivity. Direct inject mass spectrometry techniques can rarely provide all of these. Direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry, however, is certainly capable of achieving these goals. As a multifaceted tool developed in the Verbeck laboratory, many forensic applications have since been investigated (trace drug and explosives analysis). Direct inject mass spectrometry can also be easily coupled to assays to obtain additional information about the analytes in question. By performing a parallel artificial membrane assay or a cell membrane stationary phase extraction prior to direct infusion of the sample, membrane permeability data and receptor activity data can be obtained in addition to the mass spectral data that was already being collected. This is particularly useful for characterizing illicit drugs and their analogues for a biologically relevant way to schedule new psychoactive substances.

Indiana University-Purdue University Indianapolis (IUPUI) / Organic-rich substrates such as soil provide an excellent carbon source for bacteria. However, hydrocarbons such as those found in various ignitable liquids can also serve as a source of carbon to support bacterial growth. This is problematic for fire debris analysis as samples may be stored at room temperature for extended periods before they are analyzed due to case backlog. As a result, selective loss of key components due to bacterial metabolism can make identifying and classifying ignitable liquid residues by their chemical composition and boiling point range very difficult. The ultimate goal of this project is to preserve ignitable liquid residues against microbial degradation as efficiently and quickly as possible. Field and laboratory studies were conducted to monitor microbial degradation of gasoline and other ignitable liquids in soil samples. In addition to monitoring degradation in potting soil, as a worst case scenario, the effect of soil type and season were also studied. The effect of microbial action was also compared to the effect of weathering by evaporation (under nitrogen in the laboratory and by the passive headspace analysis of the glass fragments from the incendiary devices in the field studies). All studies showed that microbial degradation resulted in the significant loss of n-alkanes and lesser substituted alkylbenzenes predominantly and quickly, while more highly substituted alkanes and aromatics were not significantly affected. Additionally, the residential soil during the fall season showed the most significant loss of these compounds over the course of 30 days. To combat this problem, a chemical solution is to be immediately applied to the samples as they are collected. Various household and commercial products were tested for their efficacy at low concentrations to eliminate all living bacteria in the soil. Triclosan (2% (w/v) in NaOH) proved to be the most effective at preserving ignitable liquid residues for at least 30 days.