Using soy protein as a model and deep eutectic solvent as the extraction solvent, the effects of key factors on the extraction of soy protein based on monitoring its molecular properties were systematically studied. The conditions for the recovery of soy protein from deep eutectic solvents were explored and optimized using the response surface methodology. In addition, the influence of the deep eutectic solvent on the structure and physicochemical properties of soy protein was studied using various characterization techniques. The results showed that the hydrogen bonding interaction in the deep eutectic solvent could affect the protein extraction, and the hydrophobic interaction also played an important role in the protein extraction. Moreover, the deep eutectic solvent caused protein denaturation and other changes in the protein, thus affecting the further applications of soy proteins. This study should provide reference and guidance for green extraction of other proteins by deep eutectic solvents in laboratory or industry and provide a basis for the utilization of soy or other proteins extracted from such processes.

Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex's architecture and subunits' identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for topdown MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody−antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach.

The quaternary structure is an important feature regulating protein function. Native mass spectrometry contributes to untangling quaternary structures by preserving the integrity of protein complexes in the gas phase. Tandem mass spectrometry by collision-induced dissociation (CID) can then be used to release subunits from these intact complexes, thereby providing structural information on the stoichiometry and topology. Cumulatively, such studies have revealed the preferred release of peripheral subunits during CID. In contrast, here we describe and focus on dissociation pathways that release nonperipheral subunits from hetero-complexes in CID at high collision energies. We find that nonperipheral subunits are ejected with a high propensity, as a consequence of sequential dissociation events, upon initial removal of peripheral subunits. Alternatively, nonperipheral subunits can be released directly from a charge-reduced or an elongated intact complex. As demonstrated here for a range of protein assemblies, releasing nonperipheral subunits under controlled conditions may provide unique structural information on the stoichiometry and topology of protein complexes.