The ability to monitor protein aggregation at the molecular level is critical for progress in many areas of life sciences ranging from exploring mechanisms of amyloidosis and etiology of conformational diseases to development of safe and efficient biopharmaceutical products. Despite the spectacular progress in understanding the mechanisms of protein aggregation in recent years, many aspects of the aggregating proteins' behavior remain unclear because of the extreme difficulty in tracking evolution of these notoriously complex and heterogeneous systems. Here, we introduce an electrospray ionization mass spectrometry (ESI MS)-based methodology that allows the early stages of heat-induced aggregation to be studied by monitoring both conformational changes and formation of oligomers as a function of temperature or stress duration. The new approach allows biopolymer behavior (both reversible and irreversible processes) to be monitored in great detail over a wide temperature range. Validation of the methodology is carried out by comparing temperature profiles of model proteins and nucleic acids deduced from MS measurements and differential scanning calorimetry. In order to evaluate the suitability of ESI MS for direct profiling of soluble glycoprotein aggregates, we used heat-stressed human antithrombin, to compare size-exclusion chromatography (SEC) and ESI MS as a means to probe composition of the complex mixture of soluble oligomeric species generated by heat-induced aggregation. Once the appropriate corrections are made, the abundance of the small aggregates derived from ESI MS becomes remarkably close to that calculated based on SEC data, suggesting that ESI MS may be directly applied for at least semi-quantitative characterization of soluble protein aggregates. Application of the methodology to study heat-induced aggregation of human glucocerebrosidase and antithrombin unequivocally links loss of conformational fidelity to formation of soluble oligomers, which serve as precursors to aggregation. Sequential conformational transition of a monoclonal antibody can also be sensitively probed with this method. The ability to make a distinction between various biopolymeric species (based on the differences in their masses) and their conformers (based on the differences in their charge state distributions) allows the temperature-controlled ESI-MS measurements to be carried out in complex systems with very high degree of specificity. This unique feature of the new experimental technique makes it very appealing to the biotech and biopharmaceutical sectors, where the need to engineer/formulate stable biopolymer-based products (e.g., protein drugs) places a premium on the ability to characterize their behavior as a function of temperature with a high degree of precision and accuracy. One of the unique advantages of hydrogen/deuterium exchange mass spectrometry (HDX MS) as a tool to probe protein higher order structure and dynamics is its ability to detect distinct conformational states under certain conditions. When the exchange follows the so-called EX1 or EXX regime, a distinction among various conformers can be made based on the different levels of deuterium incorporation, which manifest themselves in the form of bi- or multi-modal isotopic distributions of protein ions. In this work we exploit this unique advantage of HDX MS and the ability of mass spectrometers to select narrow populations of protein ions to develop a method which allows structure of distinct conformers to be probed at high spatial resolution. Ubiquitin is selected as the model. Validation of the method is carried out by comparing relative magnitude of protection of individual backbone amides deduced from MS and NMR measurements. The two conformers coexisting in the model system exhibit remarkable difference in deuterium incorporation at expected portions of protein sequence. The comparison to reference conformational states of ubiquitin reveals the structural nature of these conformers. These results demonstrate the capability of the top-down HDX MS/MS to specifically capture the conformational features of individual intermediates co-existing at equilibrium.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-7140 |
| Date | 01 January 2013 |
| Creators | Wang, Guanbo |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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