Date of Award
Doctor of Philosophy
Dr. Andre Venter
Dr. David Huffman
Dr. Ekkehard Sinn
Dr. Karim Essani
Electrospray ionization mass spectrometry (ESI-MS) is one of the most well-known and versatile techniques for analyzing a broad range of molecules and it has become one of the leading techniques to study biomolecules, such as proteins. ESI-MS can accurately determine the molecular weight of proteins and provide information about their peptide sequence, post-translational modifications as well as their interaction with other molecules.
During ESI-MS analysis, by spraying a sample of proteins, prepared in form of a solution, charged droplets are produced using an electric field. As the solvent molecules gradually evaporate from these droplets, freely hovering bare protein ions remain. The ions are then sampled into the mass spectrometer where they are separated and detected based on their mass to charge (m/z) ratios.
In the recent years, a new extension of ESI-MS has been developed that allows analysis of molecules from their immediate surroundings. The technique is called desorption electrospray ionization (DESI-MS). In DESI-MS the sample preparation steps take place in close proximity to ionization step. Such features also provide the advantage of surface analysis and imaging to study spatial distribution of molecules.
While DESI shares the ionization mechanism of ESI-MS, it lacks its ability to analyze large biopolymers, and struggles to analyze proteins larger than 25kDa.
Previously our research group suggested that the loss in protein signal intensity was not due to problems with physical desorption or ionization, but rather due to incomplete protein dissolution during the desorption step.
The studies conducted in this dissertation address this shortcoming by improving protein dissolution during DESI-MS. Effect of addition of volatile ammonium salts during DESI is studied, among which ammonium bicarbonate shows significant improvement in signal to noise (S/N) ratio of proteins, specifically those with higher isoelectric points (pI). The improved S/N ratio seems to be caused by extensive removal of potassium from the protein ions. While these additives lead to improvements in the performance of DESI, their addition does not cause the same effect in ESI. The different effects of these additives in DESI and ESI are studied in terms of proteins signal intensity, S/N ratio as well as charge state distribution.
The effect of addition of the amino acid of serine to the electrospray solvent of DESI is investigated. For proteins with different molecular weights and pI values, serine shows promising improvements in the signal intensity.
Application of vaporized organic reagents in the nebulizing gas flow of the electrospray solvent of DESI is described. To add these vapors, DESI sprayer is enclosed and the vapor is delivered to the inner environment of the enclosure. By adding the vapor of ethyl acetate during DESI analysis of proteins, the attained signal intensity is increased.
Such improvements can potentially be combined during a single analysis to further better the outcome of protein analysis by DESI-MS.
Honarvar, Elahe, "Improving Protein Analysis by Desorption Electrospray Ionization (DESI-MS)" (2019). Dissertations. 3464.