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Implementation of a Model System to Probe the Relationship Between Protein Higher Order Structure and HX-MS Data Significance
Blockinger, Hayley Marie
Blockinger, Hayley Marie
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Abstract
Hydrogen exchange-mass spectrometry (HX-MS) is a method that can be used to study the higher order structure (HOS) of proteins. A protein is exposed to a deuterated solvent and amide hydrogens will exchange with deuterium depending on their solvent accessibility. The uptake in deuterium over time causes a shift in the mass of the protein, detectable by mass spectrometry. Differential HX is used to compare the uptake values between a control protein system versus an altered protein system to make inferences on what the difference in mass uptake indicates about the HOS. One such comparison group is comparing the HOS of originator biologics to biosimilars. Biologics are patented protein therapeutics that are marketed at a high cost due to the cost of clinical trials. The Patient Protection and Affordable Care Act was passed to combat the high prices of drugs. If a company can prove that a biosimilar is structurally and functionally similar to an originator biologic, then the biosimilar does not have to go through all the clinical trials like the originator biologic, reducing the cost of producing the biosimilar, therefore marketing it at an affordable cost. One area in method validation that is lacking in the HX-MS field is relating the significance of HX data to important changes in the protein HOS. To probe the relationship between HX data significance and important changes in HOS, VHH-F5 and ricin toxin subunit A (RTA) was selected as a model system. The wild type and seven single point mutations of the VHH-F5 protein were successfully expressed and ELISA was conducted to see if the point mutation had a significant impact on the relative binding affinity toward RTA, which can be related back to the HOS of the mutants. Based on the calculations by the R code developed, it was determined that four mutants had significant differences in binding affinity than the wild type– P122A, I176L, D194E, and V225A – implying that the point mutation has an impact on the HOS of the protein. Future work would determine if these significant differences are detectable by HX-MS experiments.
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Date
2022-12-31
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University of Kansas
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Keywords
Chemistry, Biology, higher order structure, mass spectrometry, protein, relative binding affinity, significance