Direct characterization of overproduced proteins by native mass spectrometry

Abstract

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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Fig. 1: An overview of the direct-MS workflow for analysis of recombinant proteins from crude samples.
Fig. 2: Types of common glycosylations and their masses.
Fig. 3: Analysis of proteins in crude samples from prokaryotic and eukaryotic expression systems.
Fig. 4: Crude samples may require dilution for efficient MS measurements.
Fig. 5: The detection limit of a protein in crude samples is ~1 µM.
Fig. 6: Direct-MS analysis of a secreted antibody unravels its multiple co-existing forms.
Fig. 7: The effect of induction time and culture volume on the analysis of recombinant proteins in crude samples.
Fig. 8: Effect of ionic strength on the spectra of a recombinant antibody.
Fig. 9: Ion mobility measurements and representative mass spectra from purified and crude samples of HSA.
Fig. 10: Effects of ammonium acetate concentration and collision energy on the spectra of the protein phosphotriesterase (PTE).
Fig. 11: Effect of resolution on the measured spectra of CBM3a.
Fig. 12: Pairwise interactions of strength and sequence mapping can be performed in crude samples.
Fig. 13: Determining RAB1A activity and interactions in crude lysates by native MS.
Fig. 14: Detailed analysis of antibody glycosylation.

Data availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

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.

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S.V., G.B.-N. and M.S. designed the experiments. S.V. and G.B.-N performed the experiments. S.V., G.B.-N. and M.S. analyzed the data. M.S., G.B.-N. and S.V. wrote and edited the manuscript.

Corresponding author

Correspondence to Michal Sharon.

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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

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