Abstract
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 ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.
Highlights
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
higher-energy collisional dissociation (HCD) outperforms ultraviolet photodissociation (UVPD) with regard to the numbers of proteoforms identified at 1% false-discovery rate (FDR), our study shows that the characterization score (C-score) distributions generated by accounting for either canonical or reduced groups of UVPD product ion types are substantially unbalanced in favor of fully characterized proteoforms, whereas in HCD experiments the fraction of proteoforms with C-score Ͼ40 is about one half or one third of the total in the case of human and mouse proteomes, respectively
In this study we have evaluated the use of 213 nm UVPD for top-down proteomics
Summary
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. Recent studies by Coon and co-workers demonstrated the potential offered by activating precursor ions using IR photons during ETD experiments (the so-called AI-ETD), and this strategy proved useful for increasing analysis throughput in TDP runs using both liquid chromatography (LC) and capillary zone electrophoresis for proteoform separation [28, 29]. This promising instrument setup is still not commercially available
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