Graduate Thesis Or Dissertation

 

From ALS to Antibodies: New Techniques in Mass Spectrometry-Based Proteoform Characterization Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/rx913x44v

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  • Altogether, this dissertation highlights the value of mass spectrometry-based analysis of intact proteins for solving problems related to understanding disease mechanisms and developing new therapeutics. Mutations to the antioxidant enzyme Cu, Zn Superoxide Dismutase (SOD1) perturb protein structure in vivo to cause 2-5% of amyotrophic lateral sclerosis (ALS) cases. Metal-deficient and disulfide-reduced immature forms of SOD1 have been implicated as toxic intermediates in ALS. A major goal for recent therapeutic interventions for ALS, therefore, is to avoid the toxicity of immature SOD1 intermediates by decreasing the abundance of immature forms or promoting maturation to the stable, metal-replete form of the enzyme. Immature forms of SOD1 have been difficult to quantify accurately by traditional approaches using western blots. However, mass spectrometry enables the direct measurement of immature forms of SOD1 from the spinal cord and brain of transgenic mouse models of ALS where human mutant SOD1 has been genetically overexpressed. The CNS-penetrant copper delivery agent Cu (II) diacetyl-bis(N4-methylthiosemicarbazonato) (“CuATSM”) has been demonstrated to increase SOD1 maturity and is the most protective compound ever tested for increasing the survival in SOD1-based mouse models of ALS. However, CuATSM is challenging to manufacture and is poorly suited for use in amorphous solid dispersants, which are important for oral drug formulation, because of its propensity to form stable crystals. The poor oral bioavailability of CuATSM requires that patients take multiple large pills several times a day to achieve the therapeutic dosage, which is a challenge for patients with swallowing difficulties. Because of the high demand for an effective therapeutic, human clinical trials were quickly organized for CuATSM without prior systematic attempts to improve CuATSM’s drug characteristics. Thus, we pursued second-generation copper-delivery drugs for ALS which improve on the limitations of CuATSM. The first part of this dissertation presents work defining the abilities of new CuATSM derivatives to deliver copper to SOD1 and other copper proteins in the central nervous system. Co-expression of the human copper chaperone for SOD1 (CCS) with mutant human SOD1G93A in mice (SOD1G93AxCCS) causes accelerated neurological deficits that are rescuable by treatment with CuATSM. Here we report that, without pharmacologic intervention, SOD1wild-typexCCS mice in our lab also exhibit a phenotype resembling motor neuron disease, featuring failure to gain weight, diminished mitochondrial Complex IV activity levels, an abundance of immature SOD1, and death in fifteen to twenty-one days, similar to our previously-published results for SOD1G93AxCCS mice. The early-development phenotype observed in SOD1wild-typexCCS mice was ameliorated by therapeutic intervention with CuATSM, as quantified by body weight increase compared to untreated animals from day six to day twenty-one. Structural derivatives of CuATSM with a phenyl ring in place of a methyl group on the di-imine ligand backbone (“phenyl-derivatives”) are easier to manufacture and more suitable for formulation in amorphous solid dispersants, therefore representing a better option for pill-based formulations if their therapeutic effects are comparable to CuATSM’s putative effects. Here we evaluated phenyl-derivatives of CuATSM for their ability to deliver copper to the CNS, using SOD1wild-typexCCS mice as a rapid-screening model. Phenyl-derivatives CuPhGlyTSM and CuPhMeTSM were at least as protective as CuATSM in SODwild-typexCCS mice, and delivered approximately as much copper to Complex IV and SOD1 as CuATSM, while additionally possessing more favorable drug characteristics. CuPhGlyTSM and CuPhMeTSM therefore warrant further investigation in the development of second-generation copper-delivery drugs for ALS. The ability to rapidly asses the entire ensemble of intact SOD1 proteoforms from tissue using mass spectrometry was critical to assessing the potential efficacy of CuATSM derivatives. However, intact mass alone is often insufficient to characterize protein structure. The past several decades have witnessed remarkable advances in the capabilities of mass spectrometry techniques, but the biggest remaining challenge is the limited ability to fragment molecules within the mass spectrometer for structural characterization. Electron-based fragmentation (ExD) is a promising solution to this problem. However, current implementations of ExD are too expensive or difficult to use. The ExD Cell technology being commercialized from Oregon State University by e-MSion, Inc. promises to fill the need for accessible electron-based fragmentation. However, specific guidance for maximizing the performance of the ExD Cell for different types of analytes has not been fully described in the existing literature. Thus, the second part of this dissertation presents work on the development of methods for using the ExD Cell to fragment different types of peptide and protein analytes. These methods were applied to confirm the amino acid sequences of model proteins carbonic anhydrase and superfolder green fluorescent protein; sequence coverages of 54% and 45%, respectively, reported here using the ExD Cell are comparable to literature values using other commercially-available ExD offerings in a denatured top-down workflow. The methods developed here represent an important step toward the more wide stream acceptance of ExD in mass spectrometry-based structural characterization of proteins. One of the most valuable applications for ExD is characterization of therapeutic monoclonal antibodies (mAbs). Therapeutic mAbs must be thoroughly characterized, but existing techniques for doing so, such as bottom-up peptide mapping, can be ambiguous and labor-intensive. ExD opens new avenues for top- and middle-down protein characterization that are faster and more comprehensive. The third part of this dissertation presents work on the use of middle-down ExD-MS to confirm the amino acid sequence of the model NIST mAb. Amino acid sequence coverage of ~50% is reported for subunits LC, Fd, and Fc/2 – comparable to previously reported values using other commercially-available ExD techniques in a middle-down workflow. Lastly, forced degradation of NIST mAb was performed and half of all oxidation sites were successfully pinpointed using the ExD Cell. The ability to map modifications in a middle-down workflow represents a significant advantage over bottom-up because of the ability to assess co-occurrence of modifications. By improving access to the powerful technique of electron-based fragmentation, the ExD Cell will help top- and middle-down workflows become more mainstream, enabling more in-depth characterization of proteoforms more quickly and with greater simplicity than possible by previous approaches.
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