Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics
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Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.
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Zhao and co-workers demonstrated the value of combining CZE separations with electron dissociation techniques for top-down analysis.
“Zhao and co-workers demonstrated the value of combining CZE separations with electron dissociation techniques for top-down analysis70.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [70]
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- Authors
- McCool, E. N., Lodge, J. M., Basharat, A. R., Liu, X., Coon, J. J., & Sun, L.
- Affiliations
- Michigan State University (US)
- University of Wisconsin–Madison (US)
- Indiana University – Purdue University Indianapolis (US)
- Indiana University School of Medicine (US)
- Corresponding author
- Sun, Liangliang
- ORCID
- Venue
- Journal of the American Society for Mass Spectrometry
- Publisher
- American Chemical Society (ACS)
- Published
- 2019-05-09
- Volume
- 30
- Issue
- 12
- Pages
- 2470-2479
- ISSN
- 1044-0305 (ISSN-L)
- PMC
- PMC6527361
- OpenAlex
- W2944207730
- Cited by
- 28
- References
- 42
- Citations by year
- 2026: 6 · 2025: 2 · 2024: 1 · 2023: 4 · 2022: 5 · 2021: 2 · 2020: 4 · 2019: 4
- FWCI
- 1.50
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Journal of the American Society for Mass Spectrometry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- IUScholarWorks (Indiana University) (submittedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM118470, R01GM125991, P41GM108538, R35GM118110)
- 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
- capillary zone electrophoresis, tandem mass spectrometry, top-down proteomics, proteoforms, Escherichia coli, size exclusion chromatography, Orbitrap Fusion Lumos, post-translational modification, N-terminal acetylation, N-terminal methionine removal, signal peptide cleavage, protein truncation
- MeSH terms
- Electron Transport, Escherichia coli, Protein Processing, Post-Translational, Electrophoresis, Capillary, Escherichia coli Proteins, Proteomics, Tandem Mass Spectrometry
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