Comparison of Top-Down Protein Fragmentation Induced by 213 and 193 nm UVPD

The growing interest in advancing tandem mass spectrometry strategies for top-down proteomics has motivated efforts to optimize ion activation strategies for intact proteins, including the comparison of 193 and 213 nm wavelengths for ultraviolet photodissociation (UVPD). The present study focuses on the performance and outcomes of UVPD for five proteins, ubiquitin, cytochrome C, frataxin, myoglobin, and carbonic anhydrase, with an emphasis on evaluating the similarities and differences in fragmentation promoted by UVPD using 193 nm versus 213 nm photons. Mass spectra were collected as a function of the number of laser pulses, and precursor depletion levels were monitored and controlled to provide consistent energy deposition between 213 and 193 nm UVPD. Fragment ions were confirmed on the basis of their isotopic distributions in m/z space to preserve both charge state and abundance information and were classified on the basis of ion type and frequency. A large portion of the total fragment ion abundance was attributable to preferential cleavages, particularly ones adjacent to proline residues. These cleavages were examined on the basis of the backbone site and abundances, revealing that a and y–2 ions N-terminal to proline residues appeared at disproportionately high abundances in 213 nm UVPD spectra as compared to 193 nm UVPD spectra, highlighting one notable difference in the top-down spectra. We theorize that these fragments are formed more efficiently in 213 nm UVPD than in 193 nm UVPD due to increased absorption of 213 nm photons at the proline amide bond.