Date of Award

9-2013

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

First Advisor

Igor A. Kaltashov

Second Advisor

Richard W. Vachet

Third Advisor

Anne B. Mason

Subject Categories

Analytical Chemistry | Chemistry

Abstract

In the last two decades, protein drugs have enjoyed a rapid growth and achieved a tremendous success in treating human diseases. However, the presence of physiological barriers greatly impedes the efficient delivery of such unconventional large molecule drugs, and therefore limits their clinical utility. An elegant way to address this challenge takes advantage of certain endogenous transporter proteins, such as human transferrin (Tf), whose ability to traverse physiological barriers has been extensively exploited. However, methods to investigate Tf-based drug delivery remained insufficient and unsatisfactory until recent development of quantitative mass spectrometry (MS). Hereby, MS-based methods have been developed and validated for quantitation of exogenous Tf in biological fluids. Particularly, different O18-labeling based approaches have been evaluated, modified and developed in this work, in order to achieve the most reliable quantitation. Alternatively, a novel approach based on indium labeling and inductively coupled plasma mass spectrometry (ICP-MS) detection has been developed for sensitive quantitation of Tf in biological fluids. The second aspect of this dissertation work focuses on the application of MS-based methods for characterization of protein drugs at different levels, ranging from protein identification, covalent structure, conformation, and interaction with physiological partners. Particularly, an O18-labeling assisted approach has been developed to identification of protein deamidation products. This new approach can readily distinguish between the two deamidated isomers. Also, an LC-MS based method has been developed for ranking the susceptibility of protein disulfide bonds to reduction, which could be applied to several disulfide bond-related analyses. Finally, a recently designed growth hormone transferrin fusion protein was studied using MS-based methods, and the molecular basis for its successful oral delivery was revealed.

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