Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Norman Chiu; submitted to the Dept. of Chemistry and Biochemistry. Title from PDF t.p. (viewed Mar. 19, 2010). Includes bibliographical references (p. 61-64).

Two popular workflows exist for quantitative proteome analysis: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), with staining to visualize proteins, and multidimensional solution phase separations of isotopically labelled peptides coupled to mass spectrometry (MS). However, the development of an alternative strategy, which combines easy-to-read differential profiling as seen in 2D-PAGE, with the sensitivity of MS for detection and identification, is needed. This thesis presents work towards the development of a workflow for high-throughput protein biomarker discovery. Multidimensional separations are vital to obtain sufficient fractionation of complex proteome mixtures. As a first dimension of separation, ion exchange chromatography (IEC) is a common choice, though it has yet to be thoroughly evaluated in terms of its effectiveness as a proteome prefractionation tool. This study used a defined set of protein standards to establish the resolution and proteome yield obtained through IEC. The evaluation uncovered significant bias in terms of protein recovery and separation. To improve throughput of a multidimensional separation strategy, a multiplexed (8-column) reversed phase liquid chromatography (RPLC) platform was constructed. The system design allowed for even distribution of flow across all columns with limited cross-loading during sample loading. This system was directly coupled to matrix-assisted laser desorption/ionization (MALDI) through a novel well plate device. The Teflon wells allowed for high recovery and no cross-contamination during collection/spotting, improved throughput, and greatly reduced the number of sample manipulation steps. An evaluation of MALDI MS, using the ThermoFisher vMALDI LTQ, for quantitative profiling was performed, employing the multiplexed LC-MALDI platform. The use of MALDI MS allowed for fast (< 5.5 hours) acquisition of quantitative data from isotopically differentiated samples partitioned over 640 fractions from two-dimensional LC. Proteins comprising 0.1% of the proteome were detected and quantified using this method. Finally, the effects of varying concentrations of acetonitrile (ACN) upon the products generated from tryptic digestions were explored. Poor enzymatic efficiency in 80% ACN was found to be responsible for an increased concentration of peptides containing missed cleavage sites. These peptides often contained unique amino acid sequences, which were not detected from complete digestions, resulting in improved protein sequence coverage following MS analysis.

Tuberculosis continues to be a major threat in public health; 8.8 million incidence of TB has been reported and 2 million deaths every year. Diagnosis of TB is impeded by slow growth of an organism with a generation time of 21 days. The emergence of multidrug-resistant TB isolates which are resistant to rifampicin and isoniazid worsened the treatment programme. Furthermore, surfacing of extensively drug-resistant TB isolates especially in HIV positive patients has led to a treatment failure. Currently available diagnostic methods are time consuming and laborious. Polymerase chain reaction-based assay proved to have a better resolution for TB strain discrimination, nevertheless are costly and cannot be routinely employed in resource poor settings. As a result there is an urgent need of cheap, cost effective and rapid diagnostic methods that will reduce a turnaround time. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry potentially offers an alternative rapid and cheaper method for discrimination of TB isolates. Proper discrimination of TB isolates depends on the sample preparation method that is capable of yielding high protein content. Conventional formic/ethanol sample preparation was investigated for mycobacteria MALDI-TOF mass spectrometric analysis. Poor quality of spectra was obtained due to a complex cell wall structure of mycobacteria which released less amounts of proteins. Further attempts were made to optimize the sample preparation method by introducing glass beads for maximum cell wall disruption. Non-consistent spectra were obtained in some mycobacterial strain; therefore it was not used as a method of choice. Introduction of delipidation step using chloroform/methanol (1:1, v/v) before formic/ethanol sample preparation step, led to a generation of reproducible and consistent spectra. This newly developed method was selected to extract protein content from large number of clinical TB isolates. With MALDI-TOF MS and chloroform/methanol-based method, all mycobacterial isolates used in the proof-of-concept were correctly identified and clustered. Fifty six of sixty clinical TB isolates were correctly identified using Biotyper software. Four were incorrectly identified; it might be possible that they carry mutations in unknown regions in their genome which led to a translation of proteins that affected the overall spectra profile. MALDI-TOF MS showed the potential to be used in the clinical laboratories for discrimination of TB isolates at lower costs. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

This dissertation presents the application of mass spectrometry to the detection and characterization of microorganisms based on biomarker identification and DNA analysis. Two major topics are covered: affinity capture mass spectrometry using immunoassay methods and methods involving insertion of membrane receptors into polymerized planar supported lipid bilayers; and the application of mass spectrometry for use in clinical microbiology for the identification of microorganisms causing bloodstream infections. Affinity capture mass spectrometry on immunoassay-based platforms studied the capture of Protein A from Staphylococcus aureus , demonstrating capture that is both selective and sensitive. Experiments illustrated successful capture from a purified source and cell lysates. Affinity capture using receptors inserted into polymerized lipid bilayers was also performed using GM1 and cholera toxin subunit B, demonstrating the enhanced stability offered by polymerizing the lipid bilayers such that direct ionization could be performed. Detection of protein binding was achieved with mass spectrometry at low molar ratios of receptor, and enzymatic digestion experiments on the protein retained at the surface illustrated the ability to characterize the protein ligand bound, lending support to using this technique for reverse pharmacological applications. Lastly, experiments demonstrated that affinity capture of surface-bound proteins can also be used to extract cells from complex mixture prior to the polymerase chain reaction, illustrating utility as a pre-treatment for detecting microorganisms in blood samples. Mass spectrometry was applied to detection of microorganisms from blood culture bottles collected from patients with bloodstream infections. Polymerase chain reaction electrospray ionization and whole cell matrix-assisted laser desorption/ionization mass spectrometry were used to characterize hematopathogens. High diagnostic accuracy was demonstrated with respect to culture-based testing and these two platforms were compared considering accuracy in identification, time to result, and cost benefit analysis. The experiments presented here cover a broad range of detection strategies for identifying proteins and microorganisms. The affinity capture techniques describe the first application of peptide capture and polymerized bilayers for mass spectrometric analysis, and the clinical mass spectrometry work demonstrates validation of two emerging techniques and the first comparative study on both platforms simultaneously. All research presented here demonstrates promise for application of mass spectrometry in diagnostic biology.

Thesis (Ph. D.)--University of Wyoming, 2005. / Title from PDF title page (viewed on March 10, 2008). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/05/28). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 155-166).

There is currently no definitive biomarker for Alzheimer's Disease (AD), confirmation of diagnosis is only possible post-mortem. Magnetic resonance spectroscopy (MRS) has potential in aiding diagnosis, an MRS scan can be performed during an MRI scan, only adding around 10 minutes to scan time. Use of data from the two scans may allow more accurate diagnosis of AD. This thesis investigates a transgenic mouse model of AD, the TASTPM mouse, using in vitro and in vivo MRS as well as matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI MS Imaging). The first aim of the study was to search for a biomarker of AD that may allow better diagnosis or further our understanding of the pathology of the disease. The second aim was to evaluate the TASTPM mouse as a model of AD for use in preclinical testing of amyloid lowering agents. The third aim was to investigate a thalamic pathology in the TASTPM mice using MALDI MS Imaging. Metabolically, we found differences between the brains of TASTPM mice and their wild type base strain in both in vitro and in vivo scans. These differences may be exploited in the preclinical testing of novel amyloid lowering therapies. We also found similarities with human AD and other mouse models, lower N-acetylaspartate, lower glutamate and higher myo-inositol are all observed in human AD, as well as the TASTPM mice in vivo. We also found further evidence of impaired neuronal energy metabolism in TASTPM mice, such as lower succinate. Cerebral hypometabolism is a symptom of human AD. The TASTPM mouse seems to be a fairly good approximation of the human disease, sharing several traits. In our investigation of the thalamic pathology, we discovered a peptide which was strongly localised to the regions of the pathology and isolated it, but were unable to identify it, the work in this area will continue.

Objectives: Fusobacteria are prominent participants in the maturation of subgingival dental plaque biofilms in humans. A number of various species belonging to the Fusobacterium genus have been recovered from the subgingival microbiota of chronic periodontitis patients. However, conventional Fusobacterium species identification is labor-intensive, time-consuming, and complicated by shortcomings in phenotypic-based classification schemes, where many fusobacteria display overlapping and non-distinguishing morphologic features and biochemical properties. In addition, molecular identification of fusobacteria is plagued with difficulties of validating the specificity of nucleic acid probes and primers to various Fusobacterium species that have closely-related interspecies genetic profiles. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and its associated analytic software, was recently approved for clinical microbiology diagnostic use by the United States Food and Drug Administration. MALDI-TOF mass spectrometry has the potential to rapidly identify cultivable clinical isolates to a species level for 4,613 different bacterial species based on mass spectra of their bacterial protein profiles, including many Fusobacterium species. The purpose of this study was to use MALDI-TOF mass spectrometry to rapidly identify the patient distribution of fusobacteria isolated from the subgingival microbiota of chronic periodontitis patients. Methods: A total of 34 chronic periodontitis patients provided 96 fresh subgingival cultivable fusobacteria isolates (one to seven isolates per patient), which were presumptively identified by their chartreuse-positive colony autofluorescence under long-wave ultraviolet light on anaerobically-incubated, non-selective, enriched Brucella blood agar primary isolation plates. Each of the presumptive fusobacteria clinical isolates were subjected to MALDI-TOF mass spectrometry analysis using a bench top mass spectrometer, Bruker FlexControl 3.0 software, and MALDI Biotyper 3.1 software (Bruker Daltonics, Billerica, MA, USA), which contains mass spectra for a variety of fusobacteria in its reference library of bacterial protein profiles. Each clinical isolate underwent on-target plate formic acid protein extraction, and was taxonomically classified with MALDI-TOF mass spectrometry within an approximately 30-45 minute time period from the point of colony harvesting from primary isolation culture plates. A MALDI Biotyper log score of equal to or larger than 1.7 was required for reliable taxonomic classification of the clinical fusobacteria isolates. Results: A majority (58.8%) of the chronic periodontitis patients yielded two or three different species of subgingival Fusobacterium on non-selective enriched Brucella blood agar primary isolation plates. Fusobacterium naviforme was identified by MALDI-TOF mass spectrometry analysis in 14 (41.2%) chronic periodontitis study patients, Fusobacterium nucleatum subspecies vincentii in 13 (38.2%) patients, Fusobacterium nucleatum subspecies polymorphum in 9 (26.5%) patients, Fusobacterium nucleatum and Fusobacterium species each in 6 (17.6%) patients, Fusobacterium nucleatum subspecies nucleatum in 4 (11.8%) patients, and Fusobacterium nucleatum subspecies animalis in 3 (8.8%) patients. Three patients additionally yielded subgingival isolates of Fusobacterium canifelinum, normally an inhabitant of the oral cavity of dogs and cats. 52 (54.2%) of the fusobacteria clinical isolates revealed MALDI Biotyper log scores of equal to or larger than 1.7, the threshold for reliable taxonomic classification, while in comparison, 44 (45.8%) had log scores less than 1.7, indicating a less reliable species identification. No other microbial species, other than one of the Fusobacterium species, was listed by the MALDI-TOF mass spectrometry analytic software as the most likely organism for the tested clinical isolates. Conclusions: These findings indicate that a variety of Fusobacterium species may be identified with MALDI-TOF mass spectrometry in the subgingival microbiota of chronic periodontitis patients. F. naviforme and F. nucleatum subspecies vincentii were the most frequently isolated subgingival fusobacteria species in the evaluated study patients. Three chronic periodontitis patients also unexpectedly revealed subgingival isolates of the animal species F. canifelinum, which is normally in the oral cavity of dogs and cats. MALDI-TOF mass spectrometry may facilitate rapid identification of cultivable fusobacteria in human subgingival dental plaque biofilms, and enhance understanding of bacterial community structure in periodontal pockets. / Oral Biology

No / N-Linked glycans were ionized from several matrices with a Shimadzu-Biotech AXIMA-QIT matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer. [M+Na]+ ions were produced from all matrices and were accompanied by varying amounts of in-source fragmentation products. The least fragmentation was produced by 2,5-dihydroxybenzoic acid and the most by -cyano-4-hydroxycinnamic acid and 6-aza-2-thiothymine. Sialic acid loss was extensive but could be prevented by formation of methyl esters. Fragmentation produced typical low-energy-type spectra dominated by ions formed by glycosidic cleavages. MSn spectra (n = 3 and 4) were used to probe the pathways leading to the major diagnostic ions. Thus, for example, an ion that was formed by loss of the core GlcNAc residues and the 3-antenna was confirmed as being formed by a B/Y rather than a C/Z mechanism. The proposed structures of several cross-ring cleavage ions were confirmed and it was shown that MS3 spectra could be obtained from as little as 10 fmol of glycan