Charge Transfer Dissociation Mass Spectrometry of Biomolecules

Recent advances in many biological disciplines are closely related to the development and application of new mass spectrometry techniques. The investigation of gas-phase ion activation techniques is one of the active research fields. Although researchers have developed a variety of ion activation techniques, they all suffer from certain intrinsic limitations---either limited in the types of fragment ions, or limited by the inefficiency with low charge-state precursor ions. Most of the ion activation techniques are not commercially available, and are at their developing stages.;As an attempt to explore new possibilities of fragmenting a gas-phase ion, charge transfer dissociation (CTD) was developed by the Jackson group. CTD is not only workable with low charge state precursor ions (1+ and 2+), but also is workable with highly charged precursor ions (4+, 5+, and 6+). For peptide analysis, CTD produces extensive backbone fragment ions, including a/x, b/y, and c/z ions. Additionally, CTD generates characteristic amino acid side-chain losses, which can complement the sequence information from backbone fragments. An interesting phenomenon of CTD reaction is that the type of predominant fragment ions shifts from a/x to c/z as the precursor charge state increases from 1+ to 3+, or more.;For intact insulin analysis, CTD enables the oxidation through a one-electron (dominant) or two-electron (minor) oxidation pathway, which increased the charge state of the intact protein by 1 or 2, respectively. Direct CTD produces a few fragment ions outside the loop defined by disulfide linkages together with charge-increased/charge-decreased species. The MS3-level CID fragmentation of the charge-increased species shows the capability of breaking disulfide linkages, thus provides enhanced structural information. Making use of the ability of being workable with 1+ precursor ions, CTD was employed to fragment phospholipids with various degree of unsaturation. CTD extensively fragments the C-C single bonds within lipid acyl chain, and provides information regarding C=C double bond location. For lipids with various head groups, CTD shows the capability of fragmenting the acyl chains to some extent, but the efficiencies are not suitable for on-line HPLC experiments at this time. CTD was also applied to structural characterization of a methylated linear oligosaccharide, generating both extensive between-ring and cross-ring cleavages. Given the similarity in radical nature between CTD and ETD, CTD was integrated into a HDX workflow to probe the gas-phase conformation of ubiquitin. CTD shows comparable performance to ETD, which demonstrated the potential of CTD in pinpointing the secondary structural elements of gas-phase proteins/peptides.;In addition to the exploration to CTD technique, some efforts were devoted to examine the fragmentation behavior of radical cations generated from metastable atom-activated dissociation (MAD) reactions. ESI-generated protonated, sodiated and potassiated POPC ions were firstly subjected to MAD reactions, and then the resulting [POPC]+• ions were further mass-selected and subjected to MS3-level CID reactions respectively. The resulting mass spectra are almost identical---independent of the first generation adducting species. Moreover, the MS3 CID experiment produced extensive fragmentation along the lipid acyl chain, providing valuable structural information associated with C=C double bond positioning