Charge Transfer Dissociation Mass Spectrometry of Biomolecules

Charge Transfer Dissociation Mass Spectrometry of Biomolecules Pengfei Li 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/chargedecreased species. The MS-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 atomactivated dissociation (MAD) reactions. ESI-generated protonated, sodiated and potassiated POPC ions were firstly subjected to MAD reactions, and then the resulting