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Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry
Published on: June 8, 2021
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Top-Down Characterization of Protein Anions Using Ultraviolet Photodissociation Mass Spectrometry
Hanlin Ren1, Jennifer S Brodbelt1
1Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.
Journal of the American Society for Mass Spectrometry
|February 24, 2026
Summary
Ultraviolet photodissociation (UVPD) advances negative-mode top-down proteomics by effectively fragmenting protein anions. This method enhances sequence coverage for acidic proteome analysis, overcoming previous limitations.
Area of Science:
- Proteomics
- Mass Spectrometry
- Analytical Chemistry
Background:
- Top-down proteomics typically uses positive-mode electrospray ionization-tandem mass spectrometry (ESI-MS/MS).
- Negative-mode analysis of the acidic proteome is challenging due to low ionization efficiency and limited fragmentation methods for protein anions.
Purpose of the Study:
- To investigate the performance of ultraviolet photodissociation (UVPD) for top-down analysis of protein anions.
- To compare UVPD with higher-energy collisional dissociation (HCD) and activated electron photodetachment (a-EPD) for protein anion fragmentation.
Main Methods:
- Utilized organic bases as additives to generate abundant, highly charged protein anions.
- Applied UVPD, HCD, and a-EPD to fragment proteins ranging from 8.6 to 47 kDa.
- Analyzed fragment ions, including charge-reduced precursor radicals and various backbone fragments (a/x, b/y, c/z).
Main Results:
- UVPD produced abundant charge-reduced precursor radicals and diverse fragment ions.
- Achieved 70-95% sequence coverage for proteins <20 kDa and 30% for 47 kDa enolase, outperforming HCD and a-EPD.
- UVPD of deprotonated proteins showed charge-state independence and uniform backbone cleavage, similar to protonated proteins.
Conclusions:
- UVPD is a viable and effective fragmentation method for negative-mode top-down proteomics.
- This technique expands the scope of top-down proteomics to include acidic proteomes and improves sequence coverage.
- UVPD offers a promising alternative for analyzing protein anions, addressing key limitations in current methodologies.
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