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Development of Non-Traditional Bioanalytical Methods for the Study of Pharmacokinetics and Protein Complexes

  • Author(s): Ferguson, Carly Nicole
  • Advisor(s): Loo, Joseph A
  • et al.
Abstract

While proteomics, mass spectrometry and their combination have seen great advancements since their inception, there is room for improvement in many areas, including increased sensitivity, specificity, and applicability. Mass spectrometry-based imaging (MSI) is a relatively new technology that has found application in the medical sciences. Spatially measuring proteins, lipids, and other biomolecules in clinically relevant tissues is a powerful tool for disease biomarker studies and for disease pathology. An improved tissue imaging method has been developed that employs a series of wash steps to decrease the complexity of the lower mass range in order to better observe tissue distribution of targeted drug molecules and their metabolites. This was previously unstudied due to the common belief that any solvent would delocalize analytes of interest; however, we have shown that attention to solubility properties of these molecules allows for non-disruptive solvent selection. This method was utilized to determine the biodistribution of small molecule read-through (SMRT) compounds that are potential treatment molecules for premature termination codon (PTC) genetic disorders. MSI experiments revealed that one compound in particular, BZ16, crossed the blood-brain barrier (BBB) and entered the cerebellum. MSI was also utilized to study the distribution of a molecular tweezer (MT) compound, CLR01, which is a potential treatment for amyloid-based diseases. After administration to animal subjects, CLR01 was observed in the ventral portion of the brain, confirming the compound's ability to cross the BBB.

One area that has not been well addressed by these newer technologies is the study of protein-ligand interactions. The development of improved ambient ionization methods for mass spectrometry provides a complementary technique for the study of small molecules and their interactions with proteins and other biomolecules. Protein-ligand binding studies are crucial in the development of drug molecules; such techniques as reactive desorption electrospray ionization (DESI) allow for the study of large proteins and protein complexes in their native states.

DESI-related work has resulted in an improved protein soft ionization method, enabling the characterization of large native protein complexes (> 150 kDa). Our reactive DESI method employs two solvent capillaries: one containing buffer to maintain native conformation, and another containing electrospray-compatible solvent to more efficiently ionize proteins at higher resolution. Additionally, we have shown that we can probe protein-ligand binding stoichiometry, selectivity, and kinetics by including a small molecule in our electrospray-compatible solvent.

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