Doctor of Philosophy (PhD)
New sampling and ionization techniques were developed using a deep-ultraviolet (DUV) laser at 193 nm wavelength for analysis of large biomolecules. A micrometer spatial resolution DUV laser ablation and capture system was constructed to sample tissue biomolecules. The spot size measurements of the focused beam showed that subcellular spots with ca. 2 μm diameter can be readily achieved with this wavelength. To demonstrate the extraction of tissue biomolecules with the DUV laser, regions of tissue sections were ablated and captured for offline proteomic and genomic analysis. The proteomic studies revealed that the tissue proteins were ejected without fragmentation and that this wavelength is about four times more efficient at extracting proteins from tissue as compared to the mid-infrared 2940 nm wavelength. Similarly, the genomic analysis of the ablated and captured tissue showed that the DNA molecules remained intact following ablation. The DUV laser was also coupled with electrospray ionization for online mass spectrometry of large biomolecules. Peptides, proteins and tissue samples were ablated with the 193 nm laser and the resulting sample plume was post-ionized with electrospray. The resulting mass spectra showed intact analyte ions without indication of photodissociation. The 193 nm laser was also used to photochemically modify amino acid side chains of proteins in the electrospray ion source. A solution of protein and hydrogen peroxide was electrosprayed at the mass spectrometer inlet and the spray was irradiated with the laser to create hydroxyl radicals that subsequently oxidized the biomolecules in the electrospray. Oxidized protein peaks were observed in the acquired mass spectra. This new technique offers a general method to rapidly perform photochemical reactions in the spray at < 1 μs time scales and can potentially be coupled with tandem mass spectrometry for structural analysis of proteins. Overall, these results demonstrate that the 193 nm wavelength laser can be utilized as a high precision sampling and ionization tool xii for biomolecular analysis without damage to the analytes. The sampling resolution achievable with this wavelength makes it a promising tool for single cell biochemical analysis.
Lawal, Oluwaremilekun Omowunmi, "Deep-Ultraviolet Laser Ablation for Biomolecular Analysis" (2020). LSU Doctoral Dissertations. 5413.
Available for download on Saturday, October 28, 2023