Proteins derived by recombinant technologies must be characterized to ensure quality, consistency and optimal production. These properties are usually assayed following purification procedures that are time consuming and labor intensive. Here, we describe a native mass spectrometry (MS) approach, direct-MS, for rapid characterization of intact overexpressed proteins immediately from crude samples. In this protocol, we discuss the multiple applications of the method and outline the necessary steps required for sample preparation, data collection and interpretation of results. We begin with the sample preparation workflows, which are relevant for recombinant proteins produced within bacteria, those analyzed straight from crude cell lysate, and secreted proteins generated in eukaryotic expression systems that are assessed directly from the growth culture medium. We continue with the mass acquisition steps that enable immediate definition of properties such as expressibility, solubility, assembly state, folding, overall structure, stability, post-translational modifications and associations with biomolecules. We demonstrate the applicability of the method by presenting the characterization of a computationally designed toxin–antitoxin heterodimer, activity and protein-interaction determination of a regulatory protein and detailed glycosylation analysis of a designed intact antibody. Overall, we describe a simple and rapid protocol that is relevant to both prokaryotic and eukaryotic expression systems and can be carried out on multiple mass spectrometers, such as Orbitrap and quadrupole time-of-flight (QTOF)-based mass spectroscopy platforms, that enable intact protein detection. The procedure takes from 30 min to several hours, from sample collection to data acquisition, depending on the depth of MS analysis.
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The datasets generated during the current study are available from the corresponding author upon reasonable request.
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We thank E. Morag and E. Bayer, for providing us with the pPICK9 plasmid for CBM3a expression, and Y. Peleg, for providing the pPICK9 plasmid for HSA expression. We are grateful to O. Kersonsky and S. Fleishman for providing us with the plasmid for the expression of MBP-PTE. We also thank S. Warszawski, A. Katz, R. Diskin and S. J. Fleishman, for providing us with the growth media containing secreted antibodies, and R. Diskin, H. Cohen-Dvashi, M. Yona and T. Unger, for providing us with the growth media containing the secreted TfR1. We are also grateful for the support of a Starting Grant from the European Research Council (ERC) (Horizon 2020/ERC grant agreement no. 636752) and for Israel Science Foundation (ISF) grant 300/17. M.S. is the Aharon and Ephraim Katzir Memorial Professorial Chair.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Michael Landreh and other anonymous reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Gan, J. et al. Anal. Chem. 89, 4398–4404 (2017): https://doi.org/10.1021/acs.analchem.7b00398
Key data used in this protocol
Ben-Nissan, G. et al. Commun. Biol. 1, 213 (2018): https://doi.org/10.1038/s42003-018-0231-3
Cveticanin, J. et al. Anal. Chem. 90, 10090–10094 (2018): https://doi.org/10.1021/acs.analchem.8b02349
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Vimer, S., Ben-Nissan, G. & Sharon, M. Direct characterization of overproduced proteins by native mass spectrometry. Nat Protoc 15, 236–265 (2020). https://doi.org/10.1038/s41596-019-0233-8