Native Capillary Electrophoresis-Mass Spectrometry for Protein analysis: Current Progress and Perspectives

Abstract

Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the protein higher order structure. The coupling with electromigrative separation techniques under native conditions enables the characterization of proteoforms and highly complex protein mixtures. In this review, we present an overview of current native CE-MS technology. First, the status of native separation conditions is described for capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE), and capillary isoelectric focusing (CIEF), as well as their chip-based formats, including essential parameters such as electrolyte composition and capillary coatings. Further, conditions required for native ESI-MS of (large) protein constructs, including instrumental parameters of QTOF and Orbitrap systems, as well as requirements for native CE-MS interfacing are presented. On this basis, methods and applications of the different modes of native CE-MS are summarized and discussed in the context of biological, medical, and biopharmaceutical questions. Finally, key achievements are highlighted and concluded, while remaining challenges are pointed out.

Mass spectrometry-intensive top-down proteomics: an update on technology advancements and biomedical applications

“…As CZE performs separation in an open tubular fused silica capillary containing no stationary phase, it can achieve higher separation efficiency and less sample loss for large proteoforms than many conventional LC techniques. [29][30][31][32][33] Additionally, CZE-MS/MS has been well recognized as a valuable technique for TDP as the nanoow CE-MS interface (i.e., 50-300 nL min −1 ) provides high sensitivity for detection. [32][33][34][35][36][37][38] The Kelleher group utilized a GELFrEE (gel-eluted liquid fraction entrapment electrophoresis)-CZE-MS/MS platform to improve the detection of large proteoforms.…”

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

“…Being both universal and selective and providing high‐sensitivity and structurally rich information on the analyzed compounds, MS represents the most powerful detection mode for CE, LC, and other separation methods. The recent developments in the area of MS detection in CE methods have been described in several reviews dealing with CE–MS in general [195–198] and with its application to peptide, protein, peptidomic, proteomic, and metabolomic analyses especially [8, 16, 44, 199–201]. Importance of MS as analytical and structure elucidation method has significantly increased in the last years due to its crucial role in the previous omics technologies [44, 199, 202].…”

“…The recent developments in the area of MS detection in CE methods have been described in several reviews dealing with CE-MS in general [195][196][197][198] and with its application to peptide, protein, peptidomic, proteomic, and metabolomic analyses especially [8,16,44,[199][200][201].…”