Large-Scale Qualitative and Quantitative Top-Down Proteomics Using Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry with Nanograms of Proteome Samples
Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has attracted attention recently for top-down proteomics because it can achieve highly efficient separation and very sensitive detection of proteins. However, separation window and sample loading volume of CZE need to be boosted for a better proteome coverage using CZE-MS/MS. Here, we present an improved CZE-MS/MS system that achieved a 180-min separation window and a 2-μL sample loading volume for top-down characterization of protein mixtures. The system obtained highly efficient separation of proteins with nearly one million theoretical plates for myoglobin and enabled baseline separation of three different proteoforms of myoglobin. The CZE-MS/MS system identified 797 ± 21 proteoforms and 258 ± 7 proteins (n = 2) from an Escherichia coli (E. coli) proteome sample in a single run with only 250 ng of proteins injected. The system still identified 449 ± 40 proteoforms and 173 ± 6 proteins (n = 2) from the E. coli sample when only 25 ng of proteins were injected per run. Single-shot CZE-MS/MS analyses of zebrafish brain cerebellum (Cb) and optic tectum (Teo) regions identified 1730 ± 196 proteoforms (n = 3) and 2024 ± 255 proteoforms (n = 3), respectively, with only 500-ng proteins loaded per run. Label-free quantitative top-down proteomics of zebrafish brain Cb and Teo regions revealed significant differences between Cb and Teo regarding the proteoform abundance. Over 700 proteoforms from 131 proteins had significantly higher abundance in Cb compared to Teo, and these proteins were highly enriched in several biological processes, including muscle contraction, glycolytic process, and mesenchyme migration.
KeywordsCapillary zone electrophoresis-tandem mass spectrometry Top-down proteomics Mass-limited samples Proteoform quantification Escherichia coli Zebrafish cerebellum Zebrafish optic tectum
We thank Prof. Heedeok Hong’s group at the Department of Chemistry of Michigan State University for kindly providing the E. coli cells for this project. We thank Prof. Jose Cibelli and Mr. Billy Poulos at the Department of Animal Science of Michigan State University for their help on collecting zebrafish brains for the project. We thank the support from the National Institute of General Medical Sciences, National Institutes of Health (NIH), through Grant R01GM118470 (X. Liu) and R01GM125991 (L. Sun and X. Liu).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no competing interest.
- 1.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
- 4.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
- 7.Anderson, L.C., DeHart, C.J., Kaiser, N.K., Fellers, R.T., Smith, D.F., Greer, J.B., LeDuc, R.D., Blakney, G.T., Thomas, P.M., Kelleher, N.L., Hendrickson, C.L.: Identification and characterization of human proteoforms by top-down LC-21 tesla FT-ICR mass spectrometry. J. Proteome Res. 16, 1087–1096 (2017)CrossRefGoogle Scholar
- 8.Schaffer, L.V., Rensvold, J.W., Shortreed, M.R., Cesnik, A.J., Jochem, A., Scalf, M., Frey, B.L., Pagliarini, D.J., Smith, L.M.: Identification and quantification of murine mitochondrial proteoforms using an integrated top-down and intact-mass strategy. J. Proteome Res. 17, 3526–3536 (2018)CrossRefGoogle Scholar
- 9.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
- 13.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
- 19.Sarg, B., Faserl, K., Kremser, L., Halfinger, B., Sebastiano, R., Lindner, H.H.: Comparing and combining capillary electrophoresis electrospray ionization mass spectrometry and nano-liquid chromatography electrospray ionization mass spectrometry for the characterization of post-translationally modified histones. Mol. Cell. Proteomics. 12, 2640–2656 (2013)CrossRefGoogle Scholar
- 22.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
- 25.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
- 26.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
- 28.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
- 30.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