Doctor of Philosophy (PhD)
This dissertation on infrared laser desorption/ionization mass spectrometry encompasses fundamental studies of the desorption/ionization process and direct-from-gel laser desorption/ionization applications. Understanding of the fundamentals behind desorption and ionization can lead to improvements in the technique and to new applications. Experiments aimed at advancing this goal are wavelength studies and two-laser infrared/ultraviolet matrix-assisted laser desorption/ionization experiments. The direct-from-gel laser desorption/ionization application in which infrared laser desorption/ionization of analytes directly ionized after gel electrophoretic separation will also be presented. Protein and peptide standards were used as test analytes and the infrared lasers used were a 10.6 micrometers carbon-dioxide laser and a mid-IR optical parametric oscillator. In the wavelength experiments, the optical parametric oscillator was tuned from 2.8 to 3.6 micrometers and the minimum laser fluence to produce a detectable ion signal (threshold fluence) was recorded. Comparison of the threshold fluence to the infrared absorption of the sample indicates that the analyte is absorbing the laser light. Scanning electron microscopy images of the sample after laser irradiation show melting and indications of explosive boiling. It is concluded from these results that ionization occurs through the sacrifice of some of the protein molecules that absorb the laser energy and act as an intrinsic matrix. In the two-laser experiments, a mixture of analyte with a laser light-absorbing matrix was deposited on the sample target and irradiated with an infrared laser, followed, after an adjustable delay, by an ultraviolet nitrogen laser. Laser fluences were attenuated below the one-laser ionization threshold and two-laser signal was obtained at delays up to several hundred microseconds. The results can be explained by infrared laser heating of the sample that leads to an enhancement of ultraviolet matrix-assisted laser desorption/ionization. Direct-from-gel laser desorption/ionization experiments used the optical parametric oscillator to ionize electrophoreticly separated biomolecules directly from conventional gel slabs and capillary gels in plastic microfluidic chips. An increase in sensitivity was found when moving to the microfluidic chip design from analyses using gel slabs. This technique shows promise for the identification of both parent and fragment masses of proteins contained in gels.
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Release the entire work immediately for access worldwide.
Little, Mark, "Infrared laser desorption/ionization mass spectrometry: fundamental and applications" (2006). LSU Doctoral Dissertations. 2690.