Antibody Sequence Analysis by Controlled Proteolysis and Ion-Ion Chemistry

Hinkle, Joshua, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Hunt, Donald, AS-Chemistry, University of Virginia

The study of proteins represents an invaluable tool for the characterization of biological systems. As proteins are one of the primary functional units of the cell, serving to transmit signals, catalyze reactions, and more, possessing the tools to effectively study them is of particular importance for studying biological systems. Antibodies in particular are a product of significant interest for in-depth characterization given the strict tolerances required when using them as antibody therapeutics. Consequently, better tools to more rapidly and thoroughly analyze these molecules are deeply needed.

Mass spectrometry has become an invaluable tool for the analysis of proteins, achieving a level of precision and sensitivity largely unrivaled by competing methods. However, despite the significant strides made in recent years, several key limitations still face modern mass spectrometric methods. Two of the major limitations still facing the proteomics community are that of complete peptide coverage following proteolytic digestion and complete fragmentation coverage following peptide dissociation. Both of these issues limit the information that can be obtained in a given experiment. To address these concerns, this dissertation presents the combination of precisely controlled digestion and ion-ion reaction strategies for improved sample preparation and fragmentation, respectively. As shown here, these results achieve significantly improve on the depth of analysis for therapeutic monoclonal antibodies, though their applicability likely extends to many other systems.

PHD (Doctor of Philosophy)
Antibody, Mass Spectrometry, Electron Transfer Dissociation, Ion Parking, Middle Down, Enzyme Reactor
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