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The Development and Applications of Soft Visible-Wavelength LDI, UV LDI, and DESI Sources for the Analyses of Biomolecules by Mass Spectrometry

West, Raymond Edward, III
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470309402

Abstract Details

2016, Doctor of Philosophy, University of Toledo, Chemistry.
Soft ionization sources are often coupled to mass spectrometers for the analyses of biomolecules due to the fact that minimal fragmentation occurs to the biomolecular ions during the process of ionization. Different types of soft ionization sources, such as matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI), and desorption electrospray ionization (DESI), play an integral role in the identification, localization, quantitation, and structural elucidation of biomolecules in various samples. However, further developments and improvements of soft ionization sources and their applications are necessary. Additionally, in order to avoid the high price or unavailability of a particular ion source, it is beneficial to design custom ion sources that enable the acquisition of accurate and reproducible mass spectra for the samples of interest. It was proposed previously that visible-wavelength MALDI may be a softer ionization technique than UV MALDI and that atmospheric pressure (AP) MALDI is advantageous to vacuum MALDI for several reasons, including the possibility of higher-throughput analyses. With this in mind, an AP visible-wavelength (532 nm) LDI source was developed and coupled to an ion trap mass spectrometer (IT-MS) in transmission geometry. This custom-designed MALDI source was then optimized and used to analyze several different oligosaccharide, peptide, and protein samples by mass spectrometry (MS), tandem MS (MS/MS), and multistage MS (MSn). This visible-wavelength MALDI-MS instrument demonstrated good limits of detection (LODs) for peptides (femtomole amounts) and oligosaccharides (picomole amounts). In addition, it seemed that AP visible-wavelength MALDI is a softer ionization technique than AP UV MALDI for the MS analysis of an ionization-labile sulfated peptide. The developed AP visible-wavelength LDI-IT-MS instrument was then utilized to analyze peptides that were fluorescently labeled on the N-terminus without the addition of an external matrix. Since the fluorophore was responsible for the absorption of the visible light and the soft ionization of the analyte, this ionization technique was named fluorophore-assisted laser desorption/ionization (FALDI)-MS. FALDI-MS provides a novel way to ionize and structurally characterize specifically labeled peptides. This technique enabled soft and selective ionization of peptides labeled with rhodamine B, and FALDI MS/MS spectra of these peptides contained only rhodamine B-containing a and b fragment ion series providing an efficient way for the peptide sequencing. To expand the concept of FALDI-MS to fluorophores absorbing UV-light, UV FALDI-MS was used to analyze fluorescently labeled peptides, proteins, an oligosaccharide, and a lipid using a commercial UV (355 nm) LDI-time-of-flight (TOF)/TOF-MS instrument. UV FALDI-MS/MS confirmed that N-terminal labeling of peptides with dyes such as rhodamine B and carboxyfluorescein (FAM) contributes to favorable formation of fluorophore-containing a and b ions upon fragmentation of fluorescently labeled peptides. In addition, a protocol for efficient labeling, purification,and analysis of fluorescently labeled peptides and proteins with coumarin was developed, facilitating the soft FALDI-MS ionization of coumarin-labeled proteins. Lastly, a custom-designed DESI source was developed and coupled to an IT-MS to study the oxidation of omega-3 fatty acids by MS and MS/MS. DESI-MS is advantageous over MALDI-MS for the analysis of low-molecular weight biomolecules on the surfaces of solid samples and requires minimal sample preparation before the analysis. DESI and ESI-MS analyses of pure omega-3 fatty acids were performed after a three-week period of sample aging at the room temperature and ambient conditions in both positive and negative ion modes. The mass spectra of the omega-3 fatty acids obtained by these two soft ionization techniques in negative ion mode were similar, but DESI-MS enabled the detection of the oxidation of the omega-3 fatty acids due to aging from the surface of a solid dog kibble. This study demonstrated that DESI-MS can be utilized for the high-throughput monitoring of the oxidation of omega-3 fatty acids in solid food samples.
Dragan Isailovic (Committee Chair)
Eric Findsen (Committee Member)
Jon Kirchhoff (Committee Member)
Kenneth Hensley (Committee Member)
219 p.
Analytical Chemistry; Chemistry
mass spectrometry; soft ionization; biomolecules; LDI-MS; DESI-MS; visible-wavelength; FALDI-MS

Recommended Citations

Citations

  • West, III, R. E. (2016). The Development and Applications of Soft Visible-Wavelength LDI, UV LDI, and DESI Sources for the Analyses of Biomolecules by Mass Spectrometry [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470309402

    APA Style (7th edition)

  • West, III, Raymond. The Development and Applications of Soft Visible-Wavelength LDI, UV LDI, and DESI Sources for the Analyses of Biomolecules by Mass Spectrometry. 2016. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470309402.

    MLA Style (8th edition)

  • West, III, Raymond. "The Development and Applications of Soft Visible-Wavelength LDI, UV LDI, and DESI Sources for the Analyses of Biomolecules by Mass Spectrometry." Doctoral dissertation, University of Toledo, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470309402

    Chicago Manual of Style (17th edition)

toledo1470309402
532
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This open access ETD is published by University of Toledo and OhioLINK.