Identifier

etd-01172013-154859

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

Matrix-assisted laser ablation desorption ionization MALDI and ion mobility (IM) MALDI mass spectrometry (MS) were used for the detection and identification of microorganisms. MALDI MS is an analytical tool that separates ions by their mass-to-charge ratio (m/z) and is routinely used for bioanalysis because of its sensitivity, selectivity, general applicability, and tolerance to impurities. Ion mobility is a gas phase technique that separates ions based on their charge and collision cross-section. In this research, MALDI-TOF MS and MALDI-IM-TOF MS analysis were conducted in parallel to assess the effectiveness of MALDI-IM-TOF MS for microorganism identification. Whole cell bacteria Escherichia coli strain W 9637 and Bacillus subtilis 6633 were prepared and analyzed using both MALDI-TOF MS and MALDI-IM-TOF MS. The signals from both analysis methods were identified using a microbial database. Vacuum ultraviolet (VUV) post-ionization MALDI-IM-TOF MS was also used and additional peaks that could not be detected using MALDI-TOF MS and MALDI-IM-TOF MS were observed from B. subtilis. MALDI MS was used in combination with mass spectral fingerprinting software for the identification of whole cell bacteria in the presence of potential environmental interferants. Whole bacteria were analyzed in the presence of fumed silica, bentonite, and pollen from Juglans nigra (black walnut) at various mass ratios. The effect of the interferants on the identifications of bacteria at the genus and species level was evaluated using the bacteria fingerprinting software MB. The results showed that correct species identification for E. coli 35218, could be determined with fumed silica, bentonite, and pollen at a mass ratio of 1:1; whereas, at the same mass ratio, with diesel particulate, only genus identification could be made. Species identification for E. aerogenes 13048 with fumed silica and pollen at a mass ratio of 1:1 was achieved. Genus identification was determined for E.aerogenes with bentonite and diesel particulate. As the mass ratio of the interferant increased, the likelihood of species identification decreased with the exception of E. aerogenes with fumed silica and pollen. Under ambient conditions, laser ablation sample transfer using a mid-infrared laser at 2.94 ƒÝm was used to ablate gram-negative E. coli 35218 and gram-positive B. cereus 11178 bacterial colony particulate from a petri dish into a solvent droplet suspended above the petri dish. The solvent droplet containing the captured material was then transferred to a nanostructured-assisted laser desorption ionization (NALDI) target for analysis on a matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometer (MS). Several peaks that were observed in the NALDI spectra of both gram-negative and gram-positive correspond to phospholipid classes, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Additional phospholipids diglycosyldiglyceride (DGDG), triacylglyceride (TAG) and a lipopeptide, which are typically found in gram-positive bacteria were observed in the NALDI spectrum of B. cereus. Using LAST NALDI, phospholipids could be identified from both bacterial species without any sample pretreatment.

Date

2013

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Murray, Kermit K.

Included in

Chemistry Commons

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