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

  • Elijah N. McCool
  • Jean M. Lodge
  • Abdul Rehman Basharat
  • Xiaowen Liu
  • Joshua J. Coon
  • Liangliang SunEmail author
Focus: Protein Post-translational Modifications: Research Article

Abstract

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.

Keywords

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

Notes

Acknowledgements

We thank Prof. Heedeok Hong’s group at Michigan State University (Department of Chemistry) for kindly providing the E. coli cells for this project. We thank the support from the National Institute of General Medical Sciences, National Institutes of Health (NIH), through Grant Nos. R01GM118470 (X. Liu), R01GM125991 (L. Sun and X. Liu), P41GM108538 (J. Coon), R35GM118110 (J. Lodge and J. Coon).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

13361_2019_2206_MOESM1_ESM.docx (2.4 mb)
ESM 1 (DOCX 2476 kb)
13361_2019_2206_MOESM2_ESM.xlsx (647 kb)
ESM 2 (XLSX 646 kb)

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Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of ChemistryMichigan State UniversityEast LansingUSA
  2. 2.Genome Center of WisconsinUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  4. 4.Department of BioHealth InformaticsIndiana University-Purdue University IndianapolisIndianapolisUSA
  5. 5.Center for Computational Biology and BioinformaticsIndiana University School of MedicineIndianapolisUSA
  6. 6.Department of Biomolecular ChemistryUniversity of Wisconsin-MadisonMadisonUSA

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