Dissertation/Thesis Abstract

Investigation of the charge state distribution effects for protein analysis in novel ionization techniques
by Sardelis, Diana Lynn, Ph.D., University of the Sciences in Philadelphia, 2013, 229; 3586940
Abstract (Summary)

Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption Ionization (MALDI) are soft ionization techniques for large biomolecules. ESI produces multiple charges which is advantageous for high resolution and mass accuracy measurements performed on instruments with mass limitations, whereas MALDI produces singlycharged ions. Solvent-Assisted Inlet Ionization (SAII) and Laserspray Ionization Inlet (LSII) are novel ionization techniques that are similar to the conventional ionization sources; however they do not require the application of an electric potential and forms ions inside a heated Atmospheric Pressure (AP)-to-vacuum inlet tube. Both of these novel techniques produce a charged envelope analogous to ESI.

Since the charge state distributions of SAII and ESI are similar, the mechanisms for analyte ion formation are proposed to be comparable. To investigate this further, we compared the effects of supercharging additives and pHs for ESI and SAII under similar conditions. The results were evaluated using a novel statistical approach.

ESI and SAII were both employed using various proteins to determine if the conformational changes with the addition of charge enhancing and reducing agents provide similar results. Furthermore, these results were verified with circular dichroism to show if the ionizable side chains on the protein are being affected by the solvent, or if the protein was denatured by the additives.

In order to improve the practicality of LSII, the sensitivity of LSII will be measured by determining the limit of detection on the Orbitrap Exactive and these measurements will be compared to a vacuum MALDI-TOF ULTRAFLEX III instrument. This baseline measurement will be used as a guide for future research aimed at improving the sensitivity of the LSII method. In addition, atomic force microscopy will be utilized to determine the topography and size of the particles in the LSII ablation process to verify the material entering the mass spectrometry orifice.

Comparisons in SAII and LSII will be made with ESI to determine detection limits and mechanistic approaches for protein conformation charge state distributions. The goal is to enhance the multiple charges by utilizing supercharging and proteindenaturing additives in the inlet ionization techniques as well as determining if the mechanisms are comparable to ESI.

Indexing (document details)
Advisor: McKee, James
Commitee:
School: University of the Sciences in Philadelphia
School Location: United States -- Pennsylvania
Source: DAI-B 75/07(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Analytical chemistry, Biochemistry
Keywords: Charge state distributions, Inlet ionization, Supercharged additives
Publication Number: 3586940
ISBN: 978-1-303-85254-1
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