Characterization of protein post-translational modifications using 193 nm ultraviolet photodissociation mass spectrometry



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Protein characterization remains the highest-impact application and one of the critical challenges for mass spectrometry. The dissertation begins with a fundamental investigation of how charging methods affect the dissociation patterns of intact proteins in low charge states when using UVPD compared to higher energy collisional dissociation (HCD). UVPD outperforms HCD in efficiently fragmenting proteins in low charge states irrespective of the charging method. Next, the utility of 193 nm ultraviolet photodissociation (UVPD) to localize protein post-translational modifications (PTMs) was explored. The isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) is a critical PTM that can significantly influence the quality and efficacy of protein-based pharmaceuticals. The ability of 193 nm UVPD to distinguish Asp and isoAsp in the protonated and deprotonated peptides was investigated. The differences in the relative abundances of fragment ions uniquely generated by UVPD were leveraged to differentiate isomeric peptides containing Asp or isoAsp and to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Ubiquitination, a PTM in which ubiquitin molecules are added to substrate proteins via iso-peptide linkage, was also studied. Multiple ubiquitin chains can be added to the N-terminus or one of the seven lysines of the ubiquitin in linear or branched chains, forming complex polyubiquitin chains with distinct biological functions. UVPD, followed by gas-phase proton transfer charge reduction (PTCR), was used to localize the linkage sites in six linearly linked tetraubiquitin standards. UVPD generates a diverse array of sequence ions, resulting in congested spectra due to the overlapping isotopic distribution of highly charged fragment ions for proteins >30 kDa. PTCR was employed to decongest the resultant spectra, facilitating the assignment of previously obscured product ions. A comprehensive literature review was also undertaken to highlight the current status and future directions in analyzing low-abundance PTMs. This review emphasizes the need for advancements in mass spectrometry-based techniques to characterize PTMs like ADP-ribosylation, tyrosine sulfation, and nitration. In the final chapter, UVPD, in conjunction with native mass spectrometry, was utilized to probe the residues modified by two novel covalent inhibitors of New Delhi metallo-β-lactamase 1 (NDM1), a class B1 broad-spectrum MBL that can bind and hydrolyze almost all β-lactam antibiotics.



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