Exploring N- and C-terminal Fragment Ion Biases in UV-Photodissociation Mass Spectrometry of Intact Proteins

193 nm UV-photodissociation (UVPD) is a powerful ion activation method in tandem mass spectrometry (MS/MS) for analyzing complex biomolecules and proteins. Sample ions are isolated by their mass-to-charge m/z ratio and exposed to pulsed UV light, causing absorption of UV photons and cleavage along the amide backbone to generate informative fragment ions. The high energy deposition of UVPD and preservation of higher-order structure and modifications makes it particularly appealing for analysis of large, modified, heterogeneous, or multimeric protein states that prove challenging for conventional tandem mass spectrometry methods. It is expected that fragment ions containing the N- and C-terminal ends of each protein ion should be produced and detected equally upon amide backbone cleavage. Data among recent 193 nm UVPD-MS analyses and other MS/MS methods (such as collision-induced dissociation) show bias in the production of N- and C-terminal ions in different m/z mass-to-charge regions of mass spectra. Owing to the development of UVPD for top-down protein analysis, this method was examined in more detail for the N-term/C-term bias and was the focus of the present study. Among a set of six proteins prepared in denaturing solution conditions to generate standard and “supercharged” charge states, fragment ion identifications from proteins with greater numbers of basic residues were biased towards N-terminal ions in lower m/z regions, while proteins with acidic residues biased C-terminus-containing fragment ions in the same region. The backbone sites of generated fragment ions showed precursor charge-state dependence on the degree of ion current bias but remained intrinsically biased towards N/C-terminal fragment ions, while explorations of the charge of fragment ions proved insufficient to provide rationale for the migration of fragment ions into different m/z regions. This promotes further study of biases in top down MS/MS analysis of proteins, particularly as applied to MS techniques attempting to resolve information from spectrally noisy yet information-rich UVPD fragment ions, like proton-transfer charge reduction and internal fragment ion assignment.