Early stage drug discovery is limited by the disjunction of function and binding assays, creating an information gap that leads to the high failure rate in hit advancement. This limitation is particularly pronounced for protein-protein interactions, whose large and shallow interfaces make it difficult to distinguish hits mechanistically. To address this, we developed a cross-linking matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) platform that integrates biochemical functional response and target binding in a single assay, thereby generating a multidimensional pharmacological profile. Using the SARS-CoV-2 RBD-ACE2 interaction and a set of 17 drug candidates for a proof-of-concept study, the platform revealed a clear difference between two inhibitors that appeared indistinguishable in conventional functional assays: one showed stronger affinity and preferential ACE2 binding, while the other showed weaker and less specific binding. These mechanistic differences were consistent with the results of a cellular antiviral assay, in which only the high-affinity inhibitor improved cell viability. This work presents a mechanism-centered, rapid screening strategy that provides early multiparameter insight, enables rational selection of high-quality leads for challenging drug targets, and is compatible with high-throughput formats.

Glycosylation plays a critical role in modulating protein structure, stability, and binding properties, yet comprehensive tools to systematically characterize these effects are scarce. Here, we integrated multiple mass spectrometry (MS) techniques, including high-resolution nanoelectrospray ionization MS (nESI-MS), cross-linking matrix-assisted laser desorption/ionization time-of-flight MS (XL-MALDI-MS), native MS, ion mobility mass spectrometry (IM-MS), together with collision-induced unfolding and a temperature-controlled nESI source to comprehensively investigate glycosylation-dependent changes in protein structural and functional properties. Applying this integrated platform to human IgG Fc, we uncovered how glycosylation alterations in hospitalized COVID-19 patients impact Fc conformation, stability, and receptor binding. nESI-MS profiling revealed a loss of core fucosylation, galactosylation, and sialylation in patient samples. These changes in glycosylation, particularly the loss of fucosylation (afucosylation), correlate with enhanced FcγRIIIa binding, a more open conformation, and reduced stability. These findings highlight glycosylation as a key factor in immune dysregulation during severe COVID-19, and demonstrate the power of integrating multiple MS techniques to uncover the structural and functional consequences of glycan variation. This integrated MS platform is broadly applicable to other glycoprotein systems, including quality control in glycoengineering and research on infectious diseases.

Glycosylation is the most common protein post-translational modification, affecting protein properties and functions. Abnormal variations in glycosylation are associated with diseases, e.g., coronavirus disease COVID-19. Matrix-assisted laser desorption/ionization mass spectrometry has been widely utilized for studying protein glycosylation, after proper purification of glycopeptides or glycans using hydrophilic interaction liquid chromatography (HILIC) or laboratory-synthesized hydrophilic materials. Here, glass wool tips were developed to enrich immunoglobulin G glycopeptides and applied in the analysis of COVID-19 patient samples as a proof-of-concept. A significant decrease in galactosylation was detected in the COVID-19 patient plasma sample compared to the reference sample. The tips developed in this work provided a cheap and simple enrichment alternative to commercial HILIC tips for studying protein glycosylation.

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.

Hypertrophic scar (HS) is characterized by an abnormal fibroblast-myofibroblast transformation; non-apoptosis of fibroblasts; and redundant expression of TGF-beta 1, VEGF, alpha-SMA, and collagen I/III. An HS affects patients' physical and psychological quality of life, leading to joint dysfunction and skin cancer. However, there is currently no satisfactory drug to treat this disorder. In this study, we constructed methylprednisolone sodium succinate (MPSS) encapsulated ZIF-90 (MPSS@ZIF-90) for the effective treatment of an HS. The encapsulation of MPSS in ZIF-90 can achieve the controllable drug release of MPSS and prolong its effective treatment time. MPSS@ZIF-90 enhanced the apoptosis of human hypertrophic scar fibroblasts and downregulated the overexpression of TGF-beta 1, VEGF, alpha-SMA, and collagen I/III both in vitro and in vivo. The instant injection of MPSS@ZIF-90 effectively intervened with the formation of the HS after 28 days. On the contrary, MPSS@ZIF-90 greatly reduced the HS with two injections and 14 days of treatment after the HS was formed. This work provides evidence of effective intervention in the formation of an HS and the therapeutic effectiveness of MPSS@ZIF-90 with short treatment periods in vivo. It suggests that MPSS@ZIF-90 can be used as a biomedical option in the treatment of skin wounds and may reveal the potential molecular basis for promising future antifibrotic agents against scarring.

Proteins, and more specifically glycoproteins, have been widely used as biomarkers, e.g., to monitor disease states. Bottom-up approaches based on mass spectrometry (MS) are techniques commonly utilized in glycoproteomics, involving protein digestion and glycopeptide enrichment. Here, a dual function polymeric thiol-ene-based microfluidic chip (TE microchip) was applied for the analysis of the proteins osteopontin (OPN) and immunoglobulin G (IgG), which have important roles in autoimmune diseases, in inflammatory diseases, and in coronavirus disease 2019 (COVID-19). TE microchips with larger internal surface features immobilized with trypsin were successfully utilized for OPN digestion, providing rapid and efficient digestion with a residence time of a few seconds. Furthermore, TE microchips surface-modified with ascorbic acid linker (TEA microchip) have been successfully utilized for IgG glycopeptide enrichment. To illustrate the use of the chips for more complex samples, they were applied to enrich IgG glycopeptides from human serum samples with antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The dual functional TE microchips could provide high throughput for online protein digestion and glycopeptide enrichment, showing great promise for future extended applications in proteomics and the study of related diseases.

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) based on micro/nanostructured materials with different natures has received increasing attention for the analysis of a wide variety of analytes. However, up to now, only a few studies have shown the application of simple platforms in MALDI-MS for the identification of intact proteins. The present work reports on the application of copper oxide particles (Cu 2 O PS), obtained by a greener route, in combination with low amounts of 2,5-dihydroxybenzoic acid (DHB) as a novel hybrid platform. The combined Cu 2 O PS@DHB matrix, containing only 2.5 mg mL −1 of particles and 10 mg mL −1 of DHB, was easily applicable in MALDI-MS without surface modification of target plates. Under optimal conditions, the analysis of intact proteins up to 150,000 Da was possible, including immunoglobulin G, bovine serum albumin, and cytochrome C with adequate spot-to-spot signal reproducibility (RSD < 10%). In addition, the analysis of glycopeptides from IgG digests was carried out to prove the multipurpose application of the Cu 2 O PS@DHB platform in the low m/z range (2500–3000 Da). From the obtained results, it can be concluded that the optical and surface properties of as-synthesized Cu 2 O PS are likely to be responsible for the superior performance of Cu 2 O PS@DHB in comparison with conventional matrices. In this sense, the proposed user-friendly methodology opens up the prospect for possible implementation in bioanalysis and diagnostic research.

Graphical Abstract

As one of the most important families of porous materials, metal-organic frameworks (MOFs) have well-defined atomic structures. This provides ideal models for investigating and understanding the relationships between structures and catalytic activities at the molecular level. However, the active sites on the edges of two-dimensional (2D) MOFs have rarely been studied, as they are less exposed to the surfaces. Here, for the first time, we synthesized and observed that the 2D layers could align perpendicular to the surface of a 2D zeolitic imidazolate framework L (ZIF-L) with a leaf-like morphology. Owing to this unique orientation, the active sites on the edges of the 2D crystal structure could mostly be exposed to the surfaces. Interestingly, when another layer of ZIF-L-Co was grown heteroepitaxially onto ZIF-L-Zn (ZIF-L-Zn@ZIF-L-Co), the two layers shared a common b axis but rotated by 90 degrees in the ac plane. This demonstrated that we could control exposed facets of the 2D MOFs. The ZIF-L-Co with more exposed edge active sites exhibited high electrocatalytic activity for oxygen reduction reaction. This work provides a new concept of designing unique oriented layers in 2D MOFs to expose more edge-active sites for efficient electrocatalysis. [GRAPHICS] .

Current concerns on material-design induced mass transfer processes during small molecule electrocatalysis are on the ones assisted by external forced convection generally via electrode rotating, demonstrating the intrinsic activity of catalysts. Of note is that, in practical battery configurations, there is no the forced convection around electrode micro-environments. Therefore, the establishment of effective strategies in tuning the inherent mass transfer process, the one with no assistance by external forced convection, is also greatly significant, but rarely reported, retarding further advances. Herein, a size-induced inherent mass-transfer strategy is scrupulously established through designed kinetic investigations and also controllable construction of uniform Co, N co-doped carbon materials with a wide range of tunable particle sizes from 10 nm to 2 μm. The catalysts are synthesized by a pyrolysis of zeolitic imidazolate framework (ZIF) 67@ZIF-8, in which the wrapped shell layer avoids evident metal aggregations, and also contributes to rich porous environments after carbonizations. It is unclosed that particle size has a considerable effect on inherent mass transfer processes, even for the porous carbon catalysts. A particle size at around 700 nm is revealed to be most favorable for the inherent mass transfer process within the probed range, revealed by the smallest difference of Tafel slopes obtained with no electrode rotation and with infinite rotation speed. The latter is achieved via extrapolating rotation speeds to infinity in the Koutecký-Levich plots, by which the external mass transfer limitation can be completely eliminated. Contributed by the great inherent mass transfer process, the catalyst with a particle size of around 700 nm exhibits an impressive ORR activity in both three-electrode systems and zinc-air batteries. This work not only establishes a novel strategy in tuning inherent mass transfer process for small molecule electrocatalysis, more importantly, it provides a new dimension in kinetic investigations and oriented design of advanced energy materials.

Hearing loss is a common disease due to sensory loss caused by the diseases in the inner ear. The development of delivery systems for inner ear disease therapy is important to achieve high efficiency and reduce side effects. Currently, traditional drug delivery systems exhibit the potential to be used for inner ear disease therapy, but there are still some drawbacks. As nanotechnology is developing these years, one of the solutions is to develop nanoparticle-based delivery systems for inner ear disease therapy. Various nanoparticles, such as soft material and inorganic-based nanoparticles, have been designed, tested, and showed controlled delivery of drugs, improved targeting property to specific cells, and reduced systemic side effects. In this review, we summarized recent progress in nanocarriers for inner ear disease therapy. This review provides useful information on developing promising nanocarriers for the efficient treatment of inner ear diseases and for further clinical applications for inner ear disease therapy.