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Author ORCID Identifier

https://orcid.org/0000-0001-5183-058X

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2020

Month Degree Awarded

February

First Advisor

Igor A. Kaltashov

Subject Categories

Analytical Chemistry | Biochemistry | Molecular Biology

Abstract

Protein-based therapeutics have emerged as a key driver of rapid growth in drug development pipelines. However, developing such protein drugs is not straightforward in most cases, the existence of physiological barriers greatly restricts the efficient delivery of many therapeutic molecules, and therefore limits their clinical applications. A promising way to address this challenge takes advantage of certain transport protein which can effectively across and enhance the permeability of these barriers, such as transferrin (Tf) which can be internalized by malignant cells and cross physiological barriers via transferrin receptor (TfR)-mediated endocytosis and transcytosis. However, developing such products is impossible without successfully understanding the molecular mechanisms governing Tf/TfR interactions and the ability to monitor the biodistribution of Tf. In this work, hydrogen/deuterium exchange mass spectrometry (HDX MS) is used to investigate TfR higher order structural and dynamic changes in different Tf/TfR models that mimic various stages encountered during endocytosis. Detailed characterizations of TfR gained by HDX MS reveal the regions located at the interdomain cleft exhibiting bimodal exchange patterns may be responsible for the loss of its enzyme function in the molecular evolution. At neutral pH, a movement at the TfR/TfR interface helps to stabilize the holoTf/TfR complexation. At acidic pH, the pH-induced conformational changes at the TfR helical domain trigger a series of movements that lead to specific binding properties for holo- and apoTf C-lobe. Obtaining this information greatly enhances our understanding of the pH-dependent Tf binding properties and how TfR facilitates iron release at acidic pH. Another aspect of this dissertation work is utilizing the ability of Tf to bind to noncognate metals to trace the biodistribution of Tf. Particularly, indium has been evaluated and demonstrated as an ideal tracer of exogenous Tf in complex biological matrices using inductively coupled plasma mass spectrometry (ICP MS) as a detection tool. In addition, combining laser ablation (LA) with ICP MS detection allows distribution of exogenous Tf to be mapped within animal tissue cross-sections. The high sensitivity and selectivity of this novel approach make it an ideal quantitation/imaging tool for in vivo studies of biodistribution of Tf and Tf-based therapeutics.

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