Unique Biological Applications of Charge Transfer Dissociation Mass Spectrometry

Abstract

The ever-changing landscape of mass spectrometry is driven by gaps in knowledge, and one of the biggest driving forces is the desire to understand the structure and dynamics of biomolecules in living organisms. New and better methods of tandem mass spectrometry are often at the heart of technological developments. Some fragmentation methods are limited to expensive FTICR instruments, some techniques are limited multiply charged precursors, and others are limited to breaking only the weakest bonds in precursor ions; all of which support the pursuit of new activation methods. Charge transfer dissociation (CTD) addresses some of these concerns by providing structurally informative fragmentation of singly or multiply charged precursors using a benchtop instrument. Since its development, CTD has been successfully applied to several classes of molecules including peptides, lipids, and oligosaccharides. This work builds on those foundations to address three areas of potential interest: 1) the characterization of macrocyclic structures like antibacterial macrolides, 2) the differentiation of leucine and isoleucine residues in peptides, and 3) the discrimination of amino acid epimers and isomers, like D- and L forms of aspartic acid and isoaspartic acid in different peptides. For macrocyclic structures, we applied CTD to a variety of natural and synthetic macrocycles, including cobalamins (e.g., Vitamin B12), macrolides (e.g., Erythromycin) and a synthetic polymer (e.g., cyclic Nylon-6,6). For vitamin B12, CTD generated rich spectra that contained a variety of cleavages around the nucleotide loop but not within the corrin ring of vitamin B12. CTD of vitamin B12 also provided several neutral losses that corresponded to the axial ligand plus either the central cobalt or an acetamide neutral, which have the same nominal mass. The resolution of the 3D ion trap was insufficient to resolve the elementally distinct product ions, so some peak assignments are currently ambiguous. For Erythromycin, we again observed more numerous fragments with CTD than with CID, the latter of which was dominated by successive losses of water. Additionally, the fragments obtained with CTD were more structurally informative and indicative of the radical fragmentation produced in high energy techniques like EID and XUV-DPI by others. CTD of singly protonated Nylon-6,6 produced an abundant CTnoD oxidation peak with a charge of 2+, but no fragments. CID of the isolated 2+ radical at the MS3 level provided a rich spectrum with excellent coverage of the polymer sequence. In contrast, CID generated only successive monomer losses and water losses. For leucine and isoleucine