Abstract
The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.
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Data availability
The raw MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD047341 (refs. 72,73). The protein sequences can be accessed by the respective identifier (Supplementary Table 4) via https://www.uniprot.org/.
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Acknowledgements
We are grateful to all members of the Board of Directors of the CTDP for valuable discussions. We also thank B. Krichel and S. McIlwain (University of Wisconsin-Madison) and C. Hake (TU Darmstadt) for assistance with database searching. The CTDP, a 501c3 nonprofit corporation, is grateful for the generous support from Seer, ThermoFisher Scientific, Bruker, SCIEX, Lilly, Pfizer, Newomics and Agilent. M.T.M. thanks A. Makarov and K. Fort at ThermoFisher Scientific for support on the UHMR Q Exactive HF instrument. This work was supported by National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) grant R35 GM128624 to M.T.M, NIH grant R01 GM125085 and S10 OD018475 to Y.G. and NIH/NIGMS grant P41 GM108569 to N.L.K., as well as NIH grant R01GM103479, R35GM145286, S10RR028893 and US Department of Energy grant DEFC02-02ER63421 to J.A.L. We acknowledge project award (10.46936/intm.proj.2019.51141/60006696) from the Environmental Molecular Sciences Laboratory, a Department of Energy Office of Science User Facility sponsored by the Biological and Environmental Research program under Contract No. DE-AC05-76RL01830 to M.Z. and J.W.W. and the Ruth L. Kirschstein National Research Service Award Program at NIH (GM007185) to C.L. We are grateful for funding by the Hessian Ministry for Science and Arts via the LOEWE project ‘TRABITA’ and by the Deutsche Forschungsgemeinschaft (grant nos. 461372424 and 524226614) to F.L. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
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F.L., M.Z., B.D.S., J.A.L, C.L. and K.A.B. conceptualized the study. F.L., M.Z., B.D.S., C.L., K.A.B., Y.G., L.P.-T., W.S., J.A.L., J.N.A., N.L.K., M.T.M., H.L. and P.O.D. developed the methodology. T.H., K.A.B., B.D.S., C.L., M.Z., M.d.A.H., N.A., W.J., J.E.K., M.V., J.W.W. and Y.Y. performed the investigations. F.L. and T.H. performed formal analysis. F.L. and T.H. wrote the original draft of the manuscript. F.L., T.H., Y.G., L.P.-T., W.S., J.A.L., J.N.A., N.L.K., M.T.M., H.L. and P.O.D. contributed to the writing, review and editing of the final manuscript.
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N.L.K. is involved in entrepreneurial activities in top-down proteomics and consults for ThermoFisher Scientific. P.O.D. is the founder and principal of Eastwoods Consulting, providing business advisory services to life science companies. The remaining authors declare no competing interests.
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Habeck, T., Brown, K.A., Des Soye, B. et al. Top-down mass spectrometry of native proteoforms and their complexes: a community study. Nat Methods (2024). https://doi.org/10.1038/s41592-024-02279-6
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DOI: https://doi.org/10.1038/s41592-024-02279-6