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Studying protein structure and function by native separation–mass spectrometry

Nature Reviews Chemistry volume 6, pages 215–231 (2022)Cite this article

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Abstract

Alterations in protein structure may have profound effects on biological function. Analytical techniques that permit characterization of proteins while maintaining their conformational and functional state are crucial for studying changes in the higher order structure of proteins and for establishing structure–function relationships. Coupling of native protein separations with mass spectrometry is emerging rapidly as a powerful approach to study these aspects in a reliable, fast and straightforward way. This Review presents the available native separation modes for proteins, covers practical considerations on the hyphenation of these separations with mass spectrometry and highlights the involvement of affinity-based separations to simultaneously obtain structural and functional information of proteins. The impact of these approaches is emphasized by selected applications addressing biomedical and biopharmaceutical research questions.

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Fig. 1: Representation of the implications of denaturing and native protein analysis exemplified by a protein and its non-covalent dimer.
Fig. 2: Hyphenation of native protein separations with mass spectrometry when using volatile or non-volatile eluents.
Fig. 3: Scope of native protein separation–MS techniques for the assessment of proteoform heterogeneity.
Fig. 4: Schematic representation of the concept of affinity capillary electrophoresis coupled to mass spectrometry.
Fig. 5: Example of top-down native CE hyphenated with MS for the characterization of Escherichia coli ribosomal proteins and protein complexes.

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Acknowledgements

This work was supported by the Netherlands Organization for Scientific Research (NWO; SATIN project, grant no. 731.017.202) and the LUMC Fellowship 2020 (E.D.-V.).

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Authors and Affiliations

  1. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands

    Guusje van Schaick, Manfred Wuhrer & Elena Domínguez-Vega

  2. Division of Bioanalytical Chemistry, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, Netherlands

    Rob Haselberg & Govert W. Somsen

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  1. Guusje van Schaick
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  2. Rob Haselberg
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  3. Govert W. Somsen
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  4. Manfred Wuhrer
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  5. Elena Domínguez-Vega
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Contributions

G.v.S. researched data for the article, contributed to discussion of content and writing. E.D.-V. contributed to discussion of content, writing and reviewing/editing the manuscript before submission. R.H., G.W.S. and M.W. contributed to the reviewing and editing of the manuscript before submission.

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Correspondence to Elena Domínguez-Vega.

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Glossary

Post-translational modifications

Reversible or irreversible chemical changes of proteins occurring after protein biosynthesis.

Proteoforms

Different molecular forms of a protein derived from a single gene, including changes due to genetic variations, alternatively spliced RNA transcripts and post-translational modifications.

Bottom-up

Mass spectrometry approach for protein characterization that relies on the use of (enzymatic) cleavage of proteins into peptides prior to (liquid chromatography) tandem mass spectrometry analysis.

Top-down

Mass spectrometry approach for protein identification based on gas-phase fragmentation of intact protein ions.

Higher order structure

(HOS). The overall tertiary and quaternary structure of a protein, including folding, spatial conformation, complexation and aggregation.

Native mass spectrometry

(nMS). Mass spectrometry approach for the analysis of proteins and protein complexes while largely preserving their native and higher order structure.

Electrospray ionization

(ESI). Spray ionization process in which molecules in solution are ionized and transferred to the gas phase via formation and desolvation of highly charged droplets that are formed by application of a potential difference.

Ionization suppression

The reduced ionization efficiency of a molecule due to the presence of an interfering species, such as buffers or salts.

Post-column flow splitter

In liquid chromatography–mass spectrometry, a device installed after the separation column that splits the column effluent to achieve a reduced flow rate for enhanced electrospray ionization prior to mass spectrometry detection.

Membrane ion suppressors

In liquid chromatography–mass spectrometry, a device that can be used between the separation column and the electrospray source to remove non-mass-spectrometry-compatible components prior to ionization.

Charge-state distributions

In mass spectrometry, the various ions with different m/z values observed for one protein after electrospray ionization.

Mass-to-charge ratios m/z

In mass spectrometry, ions are separated according the ratio of their mass m (in atomic mass units) to their charge z, potentially providing information on the molecular mass upon deconvolution. Each ion of the same species with a different m/z is commonly denoted as charge state.

Ion desolvation

The removal of solvent molecules clustered around gas-phase ions by, for example, heating and/or collisions with gas molecules.

Mass resolution

The ability of a mass spectrometer to separate ions with different m/z values.

Complex-down

In mass spectrometry, the use of ion activation to eject a monomer or ligand from a biomolecular complex while inducing significant cleavage of covalent bonds, providing information on the sequence of the species.

Isocratic

Type of liquid chromatographic separation where the mobile phase has a constant composition throughout the entire analysis.

Monoclonal antibodies

(mAbs). Antibodies pool with a single, shared polypeptide sequence, often produced recombinantly in a mammalian cell line.

Gradient elution

Type of chromatographic separation where the mobile phase composition is changed during the analysis according a preset scheme.

2D LC

An analytical approach in which samples are analysed by two sequential liquid chromatography (LC) modes in order to achieve higher separation power.

Electrophoretic mobility

The specific rate of migration of charged analytes in a solution upon application of an electric field.

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van Schaick, G., Haselberg, R., Somsen, G.W. et al. Studying protein structure and function by native separation–mass spectrometry. Nat Rev Chem 6, 215–231 (2022). https://doi.org/10.1038/s41570-021-00353-7

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