Analysis of Protamines and the Investigation of Concentration Effects of an Immobilized Aspergillopepsin I Reactor

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D'Ippolito, Robert, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Hunt, Donald, AS-Chemistry, University of Virginia

The following dissertation details three projects utilizing mass spectrometric methods for protein analysis. Each project presented different challenges that required diverse methodology for successful analysis.
The first project identified protamines, which are small arginine rich proteins, from the common liverwort, Marchantia polymorpha. Protamines are essential for the condensation of DNA in spermatozoa. The origins of protamines remain elusive. Preliminary evidence detail that protamines evolved from histone H1 through a protamine-like intermediate. However, their evolutionary ancestry has only been loosely related to H1. This work provided definitive evidence linking protamines to their H1 ancestors. Additionally, a novel post translational modification was discovered. The C-termini of protamines were modified with the addition of di-aminopropane.
The second project further developed a novel protein digestion technique utilizing the nonspecific protease Aspergillopepsin I. The protease was immobilized to solid support, enabling digestion on the timescale of seconds to generate hundreds of overlapping peptides. These peptides can be further used to provide unambiguous primary structure analysis of a protein. However, the specific concentration of 0.2 μg/μL was needed to provide the expected results using this system. Multiple protein standards were used to fully characterize the effect of concentration, clearly showing that observed cleavages increased as starting concentration decreased. Additives were used to try to reverse these effects, which led to the discovery of protamines from salmon as an effective competitive inhibitor. Protamine treatment enabled the digestion of proteins at concentrations an order of magnitude lower than previously possible.
The final project incorporated the knowledge gained from the concentration effects of the enzyme reactor and applied them to bispecific antibody analysis. These engineered protein complexes represent a novel avenue for therapeutic molecules and require extensive characterization of their structure. Here, two different classes of bispecific antibodies were digested by the Aspergillopepsin I enzyme reactor to obtain near complete sequence coverage and extensive characterization of specific features of the molecules using novel methodologies.

PHD (Doctor of Philosophy)
Mass Spectrometry, Protamine, Di-aminopropane, Aspergillopepsin I, Bispecific Antibody, bsAb
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