Probing protein higher-order structures by native capillary electrophoresis-mass spectrometry

The coupling of chromatography with mass spectrometry (MS) has becoming a routine technique in proteomics for protein primary sequence and post translational modification determinations. Up to date, great efforts have been made to extend its capability to probe protein higher order structures (HOS) in the hope of achieving high-throughput protein structure analysis in complex samples. With complementary analytical capabilities, the coupling of native capillary electrophoresis and mass spectrometry (CE-MS) emerges as a promising approach for intact protein and protein complex conformation investigations. This review summarizes recent native CE-MS advances in two aspects: 1. native CE-MS for separation and differentiation of proteoforms; 2. native CE-MS methodology developments for protein HOS related parameter measurements, including protein effective charge, solvent accessible surface area, protein-ligand binding constant, 3D shape and dimensions. In this review, we focus on native CE-MS works with an emphasize on protein HOS information acquisition, and the developments of CE-MS interfaces or methods for top-down proteomics were not covered, which has been well summarized in literature. • In this review, we have discussed recent advances on the hyphenation of CE with MS for probing protein HOSs in native or close to their physiological conditions. • The advances in both CE and MS instrumentation enabled high throughput proteoform analyses, and growing numbers of proterforms are being identified in biological samples. • Besides distinguishing protein proteoforms, CE-nMS methods were also developed to extract protein HOS related structural parameters. • Both methodology and application developments have driven this technology forward, which helped CE-MS to obtain previously inaccessible structural information and expands its application in structural biology.