Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.
Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.
Novel Analytical Methods Combining Non-Denaturing Chromatography and Mass Spectrometry to Study Biopolymer Structure and Interactions
Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Igor A. Kaltashov
Richard W. Vachet
Paul L. Dubin
Stephen J. Eyles
Biotherapeutics, an emerging class of medicines containing biopolymers (e.g., proteins, peptide, and polysaccharides) have been developed for a variety of indications including cancer, autoimmune, genetic, and blood diseases. Among these biopolymers protein therapeutics have been the rapidly growing segment in the pharmaceutical industry. This trend combined with the complexity of proteins has necessitated the development of powerful and robust analytical methods to study their structure and interactions with physiological partners.
Mass spectrometry (MS) has become an indispensable tool to analyze various attributes of protein drugs such as profiling of intact mass, amino acid sequencing, and post translational modifications (PTMs). In this work novel analytical methods have been developed by combining non-denaturing chromatographic separations (such as size exclusion chromatography and ion exchange chromatography) with native ESI MS.
In addition to providing information of mass and non-covalent assemblies, native ESI MS has an advantage of probing conformational integrity of proteins by analyzing ionic charge state distributions in the mass spectrum. Size exclusion chromatography with online native ESI MS detection method developed in this work allowed characterization of higher order structure of proteins and probe aggregation propensity. Using the same SEC/native ESI MS workflow a method to find kinetics and equilibrium binding constant of transient protein interactions have also been developed. Combination of ion exchange chromatography (IXC) with native ESI MS and MS/MS detection was developed and shown to be effective in characterization of positional isomers of protein conjugates and PTMs of a biotherapeutic recombinant human interferon beta 1a. To characterize highly heterogeneous PEGylated glycoprotein (conjugated with a 20kDa PEG) analytical methods using IXC/native ESI MS combined with limited charge reduction and collisional activation were developed.
Muneeruddin, Khaja, "Novel Analytical Methods Combining Non-Denaturing Chromatography and Mass Spectrometry to Study Biopolymer Structure and Interactions" (2016). Doctoral Dissertations. 676.