Rapid Profiling of the Glycosylation Effects on the Binding of SARS-CoV-2 Spike Protein to Angiotensin-Converting Enzyme 2 Using MALDI-MS with High Mass Detection.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 binds directly to angiotensin-converting enzyme 2 (ACE2), mediating the host cell entry of SARS-CoV-2. Both spike protein and ACE2 are highly glycosylated, which can regulate the binding. Here, we utilized high-mass MALDI-MS with chemical cross-linking for profiling the glycosylation effects on the binding between RBD and ACE2. Overall, it was found that ACE2 glycosylation affects the binding more strongly than does RBD glycosylation. The binding affinity was improved after desialylation or partial deglycosylation (N690) of ACE2, while it decreased after degalactosylation. ACE2 can form dimers in solution, which bind more tightly to the RBD than the ACE2 monomers. The ACE2 dimerization and the binding of RBD to dimeric ACE2 can also be improved by the desialylation or deglycosylation of ACE2. Partial deglycosylation of ACE2 increased the dimerization of ACE2 and the binding affinity of RBD and ACE2 by more than a factor of 2, suggesting its high potential for neutralizing SARS-CoV-2. The method described in the work provided a simple way to analyze the protein-protein interaction without sample purification. It can be widely used for rapid profiling of glycosylation effects on protein-protein interaction for glycosylation-related diseases and the study of multiple interactions between protein and protein aggregates in a single system.

Ion Bunching in Square-Wave-Driven Mobility Capillary Electrophoresis-Mass Spectrometry.

The ion-bunching effect was typically produced for ion beams in the gas phase, such as in ion accelerators. In this work, ion bunching was generated for ions in a liquid channel, specifically in a mobility capillary electrophoresis-mass spectrometry (MCE-MS) setup. MCE was recently developed and coupled with MS for ion separation and the precise measurements of ion hydrodynamic radius and effective charge in solution. In conventional MCE, a DC high voltage is applied, which serves as the separation voltage. In this study, square waves were employed to replace this DC voltage, and the ion-bunching phenomenon was observed and characterized in both simulations and experiments. After applying a high voltage square wave, cations and anions would be bunched and concentrated at the positive and negative half cycle of the square wave, respectively. Accordingly, ion signal intensities detected by the following mass spectrometer could be increased by up to ∼50 folds for the aspartic acid anion. This square wave could also dissociate metal adduct cations from nucleic acid anions, which results in stronger nucleic acid ion intensities (up to ∼10 folds) with cleaner backgrounds.

Toward Nanopore Electrospray Mass Spectrometry: Nanopore Effects in the Analysis of Bacteria.

The shape and structure analyses capability of nanopore is powerful and complementary to mass spectrometry analysis. It is extremely attractive but challenging to integrate these two techniques. The feasibility of combining nanopore electrospray with mass spectrometry was explored in this study. A nanopore effect was observed during the nano-electrospray of single bacterium, through which the shape and dimension of a single bacterium could be obtained. Molecular information on these bacteria was then acquired by analyzing these bacteria deposited on the counter electrode through laser spray ionization mass spectrometry experiments. Proof-of-concept experiments were carried out for four types of bacteria. Results show that the combination of nanopore results with mass spectrum data could effectively improve the identification accuracy of these bacteria from 72.5% to 100%. Although initial experiments were demonstrated in this work, results showed that it is feasible and promising to integrate nanopore technology with mass spectrometry for large biomolecule studies in the near future.