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J Am Soc Mass Spectrom. 2019 May 9. doi: 10.1007/s13361-019-02206-6. [Epub ahead of print]

Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics.

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Department of Chemistry, Michigan State University, 578 S Shaw Lane, East Lansing, MI, 48824, USA.
Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI, 53706, USA.
Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, 719 Avenue, Indianapolis, IN, 46202, USA.
Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, 410 West 10th Street, Indianapolis, IN, 46202, USA.
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
Department of Chemistry, Michigan State University, 578 S Shaw Lane, East Lansing, MI, 48824, USA.


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% (nā€‰=ā€‰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.


Activated ion electron transfer dissociation; Capillary zone electrophoresis-tandem mass spectrometry; Disulfide bonds; Escherichia coli; Lysine succinylation; S-thiolation; Top-down proteomics

[Available on 2020-11-09]

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