Cited by 55Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsMicrofluidic and Capillary Electrophoresis Applications

Coupling Capillary Zone Electrophoresis with Electron Transfer Dissociation and Activated Ion Electron Transfer Dissociation for Top-Down Proteomics

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Zhao, Yimeng, Riley, Nicholas M., Sun, Liangliang, Hebert, Alexander S., Yan, Xiaojing, Westphall, Michael S., Rush, Matthew J. P., Zhu, Guijie +5 more·2015·Analytical Chemistry

Abstract

Top-down proteomics offers the potential for full protein characterization, but many challenges remain for this approach, including efficient protein separations and effective fragmentation of intact proteins. Capillary zone electrophoresis (CZE) has shown great potential for separation of intact proteins, especially for differentially modified proteoforms of the same gene product. To date, however, CZE has been used only with collision-based fragmentation methods. Here we report the first implementation of electron transfer dissociation (ETD) with online CZE separations for top-down proteomics, analyzing a mixture of four standard proteins and a complex protein mixture from the Mycobacterium marinum bacterial secretome. Using a multipurpose dissociation cell on an Orbitrap Elite system, we demonstrate that CZE is fully compatible with ETD as well as higher energy collisional dissociation (HCD), and that the two complementary fragmentation methods can be used in tandem on the electrophoretic time scale for improved protein characterization. Furthermore, we show that activated ion electron transfer dissociation (AI-ETD), a recently introduced method for enhanced ETD fragmentation, provides useful performance with CZE separations to greatly increase protein characterization. When combined with HCD, AI-ETD improved the protein sequence coverage by more than 200% for proteins from both standard and complex mixtures, highlighting the benefits electron-driven dissociation methods can add to CZE separations.

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Authors
Zhao, Y., Riley, N. M., Sun, L., Hebert, A. S., Yan, X., Westphall, M. S., Rush, M. J. P., Zhu, G., Champion, M. M., Medie, F. M., Champion, P. A. D., Coon, J. J., & Dovichi, N. J.
Affiliations
  • University of Notre Dame (US)
  • University of Wisconsin–Madison (US)
  • Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
Venue
Analytical Chemistry
Published
2015-05-06
Volume
87
Issue
10
Pages
5422-5429
ISSN
  • 0003-2700 (ISSN-L)
  • 1520-6882
OpenAlex
W2045261439
Cited by
55
References
61
Citations by year
2025: 2 · 2024: 1 · 2023: 3 · 2022: 3 · 2021: 3 · 2020: 1 · 2019: 6 · 2018: 7 · 2017: 8 · 2016: 10 · 2015: 10 · 2013: 1
FWCI
5.20
Type
journal article
Language
en
Open access
Locations
  • Analytical Chemistry (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • Division of Graduate Education (DGE-1256259)
  • National Institute of General Medical Sciences (R01GM080148, R01GM096767)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
Keywords
top-down proteomics, capillary zone electrophoresis, electron transfer dissociation, higher-energy collisional dissociation, protein sequence coverage, protein isoforms
MeSH terms
Amino Acid Sequence, Bacterial Proteins, Electron Transport, Molecular Sequence Data, Electrophoresis, Capillary, Mycobacterium marinum, Proteomics, Tandem Mass Spectrometry

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