Applications of mass spectrometry to bacterial diagnostics: Affinity capture matrix assisted laser desorption/ionization mass spectrometry and polymerase chain reaction mass spectrometry

Persistent Link:
http://hdl.handle.net/10150/305352
Title:
Applications of mass spectrometry to bacterial diagnostics: Affinity capture matrix assisted laser desorption/ionization mass spectrometry and polymerase chain reaction mass spectrometry
Author:
Kaleta, Erin
Issue Date:
2011
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
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.
Type:
text; Electronic Dissertation
Keywords:
Matrix-assisted laser desorption/ionization; polymerase chain reaction; Chemistry; affinity capture; mass spectrometry
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry and Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Wysocki, Vicki H.
Committee Chair:
Wysocki, Vicki H.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleApplications of mass spectrometry to bacterial diagnostics: Affinity capture matrix assisted laser desorption/ionization mass spectrometry and polymerase chain reaction mass spectrometryen_US
dc.creatorKaleta, Erinen_US
dc.contributor.authorKaleta, Erinen_US
dc.date.issued2011-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThis 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.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectMatrix-assisted laser desorption/ionizationen_US
dc.subjectpolymerase chain reactionen_US
dc.subjectChemistryen_US
dc.subjectaffinity captureen_US
dc.subjectmass spectrometryen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistry and Biochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWysocki, Vicki H.en_US
dc.contributor.chairWysocki, Vicki H.en_US
dc.contributor.committeememberSaavedra, S. Scotten_US
dc.contributor.committeememberWolk, Donnaen_US
dc.contributor.committeememberCordes, Matthewen_US
dc.contributor.committeememberGhosh, Indraneelen_US
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