Recent Advances in Mass Spectrometry-based Separation of Native Proteins

Abstract

Native proteins refer to proteins that exist in their natural state, have a correctly folded three-dimensional structure, and have biological functions. Characterization of protein higher-order structure and protein-protein interactions is crucial for a deeper understanding of protein structure and function, as well as drug development. Native mass spectrometry (nMS) can provide key information about the intact mass, subunit composition, stoichiometry, and post-translational modification sites of protein complexes or individual proteins. However, when directly analyzing complex mixtures, the resolution of nMS is reduced, and it becomes difficult to detect low-abundance proteins. Therefore, sample separation and purification play an important role in nMS studies of proteins. In this review, we describe the mainstream native separation methods coupled to mass spectrometry, including liquid chromatography and capillary electrophoresis, and discuss the challenges encountered when these technologies are combined with mass spectrometry and the latest advances in the characterization of native proteins. The article provides a comprehensive overview of non-denaturing separation methods, including practical application issues, such as buffer selection, flow rate control, and interface technology. At the same time, potential native separation technologies, such as gradient focusing and free-flow electrophoresis that have not been widely used in nMS are also introduced, providing new perspectives for high-resolution detection of complex samples and detection of low-abundance proteins.

Keywords

Native mass spectrometry / Protein separation / Liquid chromatography / Capillary electrophoresis / Protein structure / Protein-protein interaction / Free-flow electrophoresis / Chemical Sciences / Analytical Chemistry / Biological Sciences / Biochemistry and Cell Biology / Medical and Health Sciences / Medical Biochemistry and Metabolomics

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