Doctor of Philosophy (PhD)
The goal of this dissertation was to demonstrate collection, detection and identification of microorganisms from bioaerosols using offline matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of proteins. MALDI-MS intact bacteria techniques were adapted for use with an orthogonal MALDI quadrupole-time-of-flight mass spectrometer. Results indicate the instrument is capable of analyzing intact whole-cells. The first phase was to evaluate three bioaerosol samplers, an Andersen N6 single-stage impactor (AI), a cyclone impactor, and a vacuum filter system. The samplers collected test bioaerosols using a constructed bioaerosol exposure chamber (BEC). The BEC allowed all three samplers to operate in parallel. Each sampler demonstrated the ability to successfully collect and detect the test bioaerosol by offline MALDI-MS. Using the TOF-MS spectra from impacted bacteria, the Expert Protein Analysis System's (ExPASy) sequence retrieval system (SRS) was used to search the SWISS-PROT database. A total of 19 unique proteins were identified for E. coli,8 for B. Thuringiensis, and 6 for B. subtilis. Subsequently, cytochrome c and E. coli samples were proteolyzed in situ using trypsin and CNBr. The digestions were done using removable mini-wells. The mini-wells were placed on top of collected spots on the MALDI target and served as a mini chemical reactor for digestion. Using the TOF-MS spectra of the digested samples, peptide mass mapping was done using the MASCOT search engine. A progressive reductive iterative search mapping (PRISM) technique was used in order to assist in optimizing protein matches from E. coli. In this approach, four of seven iterations produced protein matches. To determine the suitability of MS/MS techniques for use with in situ digests, selected fragments from the cytochrome c and E. coli digests was done. MS/MS was successful for cytochrome c, but was unable to produce spectra for E. coli.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Dugas, Alton John, "Matrix-assisted laser desorption/ionization mass spectrometry for the analysis of collected bioaerosols" (2008). LSU Doctoral Dissertations. 494.