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Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry
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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.
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“For example, online size exclusion chromatography (SEC) has been used for the rapid online desalting of antibodies and protein complexes [77].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [77]
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Metadata
- Authors
- VanAernum, Z. L., Busch, F., Jones, B. J., Jia, M., Chen, Z., Boyken, S. E., Sahasrabuddhe, A., Baker, D., & Wysocki, V. H.
- Affiliations
- The Ohio State University (US)
- California Institute of Technology (US)
- University of Washington (US)
- Amgen (United States) (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Wysocki, Vicki H.
- ORCID
- Venue
- Nature Protocols
- Published
- 2020-01-31
- Volume
- 15
- Issue
- 3
- Pages
- 1132-1157
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC7203678
- OpenAlex
- W3004074111
- Cited by
- 169
- References
- 61
- Citations by year
- 2026: 21 · 2025: 19 · 2024: 36 · 2023: 26 · 2022: 23 · 2021: 24 · 2020: 17
- FWCI
- 8.59
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Nature Protocols (publishedVersion)
- ChemRxiv (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- CaltechAUTHORS (California Institute of Technology) (submittedVersion, cc-by-nc-nd)
- Royal College of Surgeons in Ireland (submittedVersion, cc-by-nc-nd)
- Funders
- U.S. Department of Health & Human Services | National Institutes of Health (P41 GM128577, S10 OD018507)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- native mass spectrometry, protein quality control, protein complexes, cell lysate, size exclusion chromatography, protein oligomerization, protein purification, tandem mass spectrometry, sample preparation
- MeSH terms
- Buffers, Chemistry Techniques, Analytical, Chromatography, Gel, Proteins, Mass Spectrometry
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