Thorough performance evaluation of 213 nm ultraviolet photodissociation for top-down proteomics

Luca Fornelli, Kristina Srzentić, Timothy K. Toby, Peter F. Doubleday, Romain Huguet, Christopher Mullen, Rafael D. Melani, Henrique dos Santos 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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

52 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)405-420
Number of pages16
JournalMolecular and Cellular Proteomics
Volume19
Issue number2
DOIs
StatePublished - 2020

Funding

* This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Grant No. P41 GM108569 for the National Resource for Translational and Developmental Proteomics (NRTDP). Several authors are involved in software commercialization and Thermo Fisher Scientific is an Industrial Collaborator of the NRTDP. The authors have declared a conflict of interest. □S This article contains supplemental Figures and Tables. ‖ To whom correspondence should be addressed: Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, IL 60208. Tel.: 847-467-4362; Fax: 847-467-3276; E-mail: [email protected]. ** Present address: Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Norman, Oklahoma 73071. ‡‡Present address: Thermo Fisher Scientific, 790 Memorial Dr Suite 2D, Cambridge, Massachusetts 02139. §§ Present address: Cour Development Labs, 8045 Lamon Ave, Skokie, Illinois 60077. ¶¶ Present address: Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, 1800 E. Paul Dirac Drive, Tallahassee, Florida 32310.

ASJC Scopus subject areas

  • Analytical Chemistry
  • Molecular Biology
  • Biochemistry

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