Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry
Abstract
It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.
Additional Information
© 2020 Springer Nature Limited. Received 02 July 2019; Accepted 06 December 2019; Published 31 January 2020. The authors wish to thank R. Viner, A. Bailey and T. Zhang (Thermo Fisher Scientific) for assistance with the non-denaturing separations, M. Marty (University of Arizona) for assistance with UniDec, M. Bern and S.J. Skilton (Protein Metrics Inc.) for assistance with Intact Mass software, B. Rivera (Phenomenex) for helpful discussions and prototype Yarra columns, S. Thornton for assistance with figure production and S. Lai for careful proofing. The VP40 plasmid was a generous gift from the Ollmann Saphire research group (Scripps Research Institute). Work in the Wysocki laboratory was supported by National Institutes of Health Grant P41 GM128577, Ohio Eminent Scholar funds and a subaward from the University of Washington, Baker laboratory. The 15 T Bruker SolariXR FT-ICR instrument was supported by NIH Award Number Grant S10 OD018507. Design and preparation of proteins and protein complexes in the Baker laboratory was supported by the Howard Hughes Medical Institute and the generosity of Eric and Wendy Schmidt by recommendation of the Schmidt Futures program. Data availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Author Contributions: Z.L.V., F.B., M.J. and V.H.W. designed and technically developed the protocol. Z.C., S.E.B. and D.B. provided inspiration for the conception of the protocol and provided valuable ideas and feedback throughout the development and optimization of the protocol. Z.L.V., F.B., B.J.J., M.J. and A.S. performed experiments. Z.L.V. and F.B. wrote the manuscript with assistance from V.H.W. All authors discussed the results and commented on the manuscript. The authors declare no competing interests.Attached Files
Accepted Version - nihms-1575748.pdf
Supplemental Material - 41596_2019_281_MOESM1_ESM.pdf
Supplemental Material - 41596_2019_281_MOESM2_ESM.pdf
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Additional details
- Alternative title
- Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry
- PMCID
- PMC7203678
- Eprint ID
- 101244
- Resolver ID
- CaltechAUTHORS:20200212-113154271
- P41 GM128577
- NIH
- Ohio Eminent Scholar Program
- University of Washington
- S10 OD018507
- NIH
- Howard Hughes Medical Institute (HHMI)
- Schmidt Futures Program
- Created
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2020-02-12Created from EPrint's datestamp field
- Updated
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2023-06-01Created from EPrint's last_modified field