Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics
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.
KeywordsCapillary zone electrophoresis-tandem mass spectrometry Activated ion electron transfer dissociation Top-down proteomics Escherichia coli S-thiolation Disulfide bonds Lysine succinylation
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.
- 3.Tran, J.C., Zamdborg, L., Ahlf, D.R., Lee, J.E., Catherman, A.D., Durbin, K.R., Tipton, J.D., Vellaichamy, A., Kellie, J.F., Li, M., Wu, C., Sweet, S.M., Early, B.P., Siuti, N., LeDuc, R.D., Compton, P.D., Thomas, P.M., Kelleher, N.L.: Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature. 480, 254–258 (2011)CrossRefGoogle Scholar
- 6.Ansong, C., Wu, S., Meng, D., Liu, X., Brewer, H.M., Deatherage Kaiser, B.L., Nakayasu, E.S., Cort, J.R., Pevzner, P., Smith, R.D., Heffron, F., Adkins, J.N., Pasa-Tolic, L.: Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella typhimurium in response to infection-like conditions. Proc. Natl. Acad. Sci. U. S. A. 110, 10153–10158 (2013)CrossRefGoogle Scholar
- 8.Roth, M.J., Plymire, D.A., Chang, A.N., Kim, J., Maresh, E.M., Larson, S.E., Patrie, S.M.: Sensitive and reproducible intact mass analysis of complex protein mixtures with superficially porous capillary reversed-phase liquid chromatography mass spectrometry. Anal. Chem. 83, 9586–9592 (2011)CrossRefGoogle Scholar
- 12.Han, X., Wang, Y., Aslanian, A., Fonslow, B., Graczyk, B., Davis, T.N., Yates 3rd, J.R.: In-line separation by capillary electrophoresis prior to analysis by top-down mass spectrometry enables sensitive characterization of protein complexes. J. Proteome Res. 13, 6078–6086 (2014)CrossRefGoogle Scholar
- 18.Sun, L., Zhu, G., Zhang, Z., Mou, S., Dovichi, N.J.: Third-generation electrokinetically pumped sheath-flow nanospray interface with improved stability and sensitivity for automated capillary zone electrophoresis-mass spectrometry analysis of complex proteome digests. J. Proteome Res. 14, 2312–2321 (2015)CrossRefGoogle Scholar
- 19.Lubeckyj, R.A., McCool, E.N., Shen, X., Kou, Q., Liu, X., Sun, L.: Single-shot top-down proteomics with capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for identification of nearly 600 Escherichia coli proteoforms. Anal. Chem. 89, 12059–12067 (2017)CrossRefGoogle Scholar
- 20.McCool, E.N., Lubeckyj, R., Shen, X., Kou, Q., Liu, X., Sun, L.: Large-scale top-down proteomics using capillary zone electrophoresis tandem mass spectrometry. J. Vis. Exp. 140, e58644 (2018)Google Scholar
- 21.Zhu, G., Sun, L., Dovichi, N.J.: Thermally-initiated free radical polymerization for reproducible production of stable linear polyacrylamide coated capillaries, and their application to proteomic analysis using capillary zone electrophoresis-mass spectrometry. Talanta. 146, 839–843 (2016)CrossRefGoogle Scholar
- 25.Haverland, N.A., Skinner, O.S., Fellers, R.T., Tariq, A.A., Early, B.P., LeDuc, R.D., Fornelli, L., Compton, P.D., Kelleher, N.L.: Defining gas-phase fragmentation propensities of intact proteins during native top-down mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 1203–1215 (2017)CrossRefGoogle Scholar
- 30.Riley, N.M., Sikora, J.W., Seckler, H.S., Greer, J.B., Fellers, R.T., LeDuc, R.D., Westphall, M.S., Thomas, P.M., Kelleher, N.L., Coon, J.J.: The value of activated ion electron transfer dissociation for high-throughput top-down characterization of intact proteins. Anal. Chem. 90, 8553–8560 (2018)CrossRefGoogle Scholar
- 31.Zhao, Y., Riley, N.M., Sun, L., Hebert, A.S., Yan, X., Westphall, M.S., Rush, M.J., Zhu, G., Champion, M.M., Mba Medie, F., Champion, P.A., Coon, J.J., Dovichi, N.J.: Coupling capillary zone electrophoresis with electron transfer dissociation and activated ion electron transfer dissociation for top-down proteomics. Anal. Chem. 87, 5422–5429 (2015)CrossRefGoogle Scholar
- 34.Kou, Q., Xun, L., Liu, X.: TopPIC: a software tool for top-down mass spectrometry-based proteoform identification and characterization. Bioinformatics. 32, 3495–3497 (2016)Google Scholar