Capillary electrophoresis‐mass spectrometry for protein analyses under native conditions: 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 electromigration 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.

Affinity-Resolved Size Exclusion Chromatography Coupled to Mass Spectrometry: A Novel Tool to Study the Attribute-and-Function Relationship in Therapeutic Monoclonal Antibodies

“…Collectively, the findings from these two studies were highly consistent with those previously documented, highlighting the crucial roles of both macro- and microheterogeneity of Fc N-glycosylation in FcγRIIIa binding. Importantly, compared to other established workflows, such as AC-MS and ACE-MS, this new method provided a few unique advantages. For example, unlike AC-MS, the described workflow does not depend on the availability of specific affinity columns.…”

Proteomics—The State of the Field: The Definition and Analysis of Proteomes Should Be Based in Reality, Not Convenience

“…In MSi-TDP, the LC co-elution of proteoforms with multiple overlapping charge states reduces sensitivity as molecules of the same proteoform are spread across multiple charge states, yielding data that are challenging to interpret [ 60 , 184 ]. Capillary electrophoresis has the potential to completely resolve individual proteoforms from others, resulting in simplified MS spectra, but the incompatibility of solvents and buffers with ESI and the extremely small injection volumes required have restrained its routine implementation [ 185 , 186 ]. Additionally, CE cannot address the charge state loss in sensitivity and suffers from the same limitation as BUP and other MSi-TDP approaches in that any experimental replicates must be carried out sequentially as parallel replicates are not possible.…”

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. 29–33 Additionally, CZE-MS/MS has been well recognized as a valuable technique for TDP as the nanoflow CE-MS interface ( i.e. , 50–300 nL min −1 ) provides high sensitivity for detection.…”