Pushing the limits of native MS: Online SEC-native MS for structural biology applications
Résumé
Native mass spectrometry (nMS) is now widely applied to investigate non-covalently assembled biomolecule complexes. nMS requires the use of near-neutral pH and volatile buffers to preserve the native state of proteins. However, buffer exchange into nMS-compatible solvent is usually performed manually, which results in a time-consuming and tedious process, thus appearing as a major drawback for nMS analysis. Conversely, online coupling of size exclusion chromatography (SEC) to nMS affords a fast-automated and improved desalting, but also provides an additional dimension of separation for complex protein mixtures. We illustrate here the benefits of SEC-nMS compared to manual offline desalting for the characterization of a wide variety of biological systems, ranging from multiprotein assemblies, protein–ligand and protein–nucleic acid complexes, to proteins in a detergent environment. We then highlight the potential of the coupling to further integrate ion mobility while preserving the native conformations of proteins, allowing for rapid collision cross section measurement and even collision-induced unfolding experiments. Finally, we show that online SEC coupling can also serve as the basis for multidimensional non-denaturing liquid chromatography (LC) workflows, with the SEC acting as a fast desalting device, helping to achieve first dimension LC separation in optimal chromatographic conditions while being compatible with further nMS analysis.
Domaines
Chimie
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