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Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics

2019; Elsevier BV; Volume: 19; Issue: 2 Linguagem: Inglês

10.1074/mcp.tir119.001638

1535-9484

Luca Fornelli, Kristina Srzentić, Timothy K. Toby, Peter F. Doubleday, Romain Huguet, Christopher Mullen, Rafael D. Melani, Henrique S. Seckler, Caroline J. DeHart, Chad R. Weisbrod, Kenneth R. Durbin, Joseph B. Greer, Bryan P. Early, Ryan T. Fellers, Vlad Zabrouskov, Paul M. Thomas, Philip D. Compton, Neil L. Kelleher,

Biosensors and Analytical Detection

Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an