Mass spectrometry-based methods to explore higher-order protein structure: Generating a fingerprint for biologics
28 March 2018
Stadscampus, Promotiezaal van de Grauwzusters - Lange Sint-Annastraat 7 - 2000 Antwerpen (route: UAntwerpen, Stadscampus
Organization / co-organization:
Department of Chemistry
Frank Sobott & Geert Baggerman
PhD defence Sneha CHATTERJEE - Faculty of Sience, Department of Chemistry
Therapeutic proteins have emerged as an important category of biopharmaceutical products, due to their use in the treatment of cancer, inflammation and infectious diseases. To characterise proteins, we require information on the primary to higher-order structure, as these distinct structural aspects control the general structure and basic function of the protein. Rather than maintaining their native state, proteins can undergo conformational changes, which can influence their functional aspects. Characterising the protein structure and the resulting protein function, will therefore enable further evaluation of the safety and quality of the protein as a biotherapeutic agent.
One limitation to do this is that, given their highly flexible and complex structures, there is no precise protocol yet for the complete and detailed characterisation of such large biomolecules.
Mass spectrometry (MS) is a key technique for protein characterization, providing detailed information on primary to higher-order structure.
Our goal is to develop a set of MS based methods to thoroughly characterise proteins of biotherapeutic interest, especially the higher-order protein structure. This will decide the quality control of these therapeutic proteins whose biological efficacy, safety and immunogenicity are dependent on their structures.
In this thesis, a set of MS-based methods is used to characterise proteins of biotherapeutic interest such as antibodies, antibody fragments and Fc-fusion proteins. The native MS analysis of intact proteins and protein complexes is discussed. The conformational analysis of proteins was performed using ion mobility-mass spectrometry by monitoring the size and shape of proteins. Top-down electron transfer dissociation (ETD) of antibodies was discussed to analyse solvent-exposed regions of the protein. HDX-MS was employed to locate binding interfaces within protein complexes. The FPOP analysis was applied to intact antibodies and the oxidised samples were measured with native IM-MS.
The range of structural information provided by this set of MS-based methods proves how powerful these techniques can be and how they can aid in understanding the higher-order structure of proteins.