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    Laser Electrospray Mass Spectrometry for Structural Analysis of Biomolecules

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    Genre
    Thesis/Dissertation
    Date
    2017
    Author
    Karki, Santosh
    Advisor
    Levis, Robert J.
    Committee member
    Levis, Robert J.
    Valentine, Ann M.
    Zdilla, Michael J., 1978-
    McEwen, Charles N., 1942-
    Department
    Chemistry
    Subject
    Chemistry
    Charge Reduction
    Electrospray Ionization
    Femtosecond Lasers For Mass Spectromerty
    Mass Spectrometry
    Protein Confromational Analysis
    Supercharging Reagents
    Permanent link to this record
    http://hdl.handle.net/20.500.12613/1576
    
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    DOI
    http://dx.doi.org/10.34944/dspace/1558
    Abstract
    This dissertation elucidates a greater understanding of protein folding and unfolding processes during the lifetimes of electrospray and nano-spray droplets in laser electrospray mass spectrometry (LEMS) and nano-laser electrospray mass spectrometry (nano-LEMS) measurements, respectively. The similarity in mass spectral features obtained from conventional electrospray measurements for supercharged proteins with those of LEMS measurements suggested that supercharging phenomena occurs in the electrospray droplets during the droplet desolvation process. It was observed that the laser vaporization of protein from condensed phase into the electrospray droplets containing denaturing electrospray solution and a supercharging reagent resulted in the increase in ion abundance of higher charge states in comparison with electrospray measurements. Conversely, the addition of solution additives with varying gas phase basicity in the electrospray solvent resulted in charge reduction for unfolded protein upon laser vaporization from condensed phase into the charged electrospray droplets. The extent of charge reduction and the fraction of folded protein within the electrospray droplets was found to be dependent upon both the extent of protein denaturation in the solution prior to laser vaporization and the gas phase basicity of solution additives. The ability of the LEMS technique to analyze molecules from solution with high matrix effects was established by the successful detection of protein molecules from solution with high salt concentration. Experiments with LEMS enabled the detection of a protonated protein feature as the dominating peak in the mass spectra for up to 250 mM sodium chloride while conventional electrospray resulted in predominantly salt-adducted features, with suppression of the protonated protein ions for the salt concentration of 5 mM. This dissertation also expanded upon the use of a reaction system to measure the lifetimes of laser vaporized liquid droplets coupled with electrospray and nano-spray postionization mass spectrometry. Electrospray and nanospray droplet lifetimes were measured to be 4.5±0.6 ms and 1.4±0.3 ms using LEMS and nano-LEMS measurements, respectively. Time dependent protein folding measurements using LEMS revealed intermediate states during protein folding processes which are often limited in conventional electrospray measurements where bulk solution in manipulated (change in pH) to achieve protein folding.
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