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N- and O-glycosylation modifications of proteins are closely linked to the onset and development of many diseases and have gained widespread attention as potential targets for therapy and diagnosis. However, the low abundance and low ionization efficiency of glycopeptides as well as the high heterogeneity make glycosylation analysis challenging. Here, an enrichment strategy, using Knoevenagel copolymers modified with polydopamine–adenosine (denoted as PDA–ADE@KCP), was firstly proposed for simultaneous enrichment of N- and O-glycopeptides through the synergistic effects of hydrophilic and electrostatic interactions. The adjustable charged surface and hydrophilic properties endow the material with the capability to achieve effective enrichment of intact N- and O-glycopeptides. The experimental results exhibited excellent selectivity (1 : 5000) and sensitivity (0.1 fmol μL−1) of the prepared material for N-glycopeptides from standard protein digest samples. Moreover, it was further applied to simultaneous capturing of N- and O-glycopeptides from mouse liver protein digests. Compared to the commercially available zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) material, the number of glycoproteins corresponding to all N- and O-glycopeptides enriched with PDA–ADE@KCP was much more than that with ZIC-HILIC. Furthermore, PDA–ADE@KCP captured more O-glycopeptides than ZIC-HILIC, revealing its superior performance in O-glycopeptide enrichment. All these results indicated that the strategy holds immense potential in characterizing N- and O-intact glycopeptides in the field of proteomics. [ABSTRACT FROM AUTHOR]

Despite the popularity of ion mobility spectrometry (IMS) for glycan analysis, its limited structural resolution hinders the effective separation of many glycan isomers. This leads to the overlap of IMS peaks, consequently impacting the accurate identification of glycan compositions. To this end, an improved algorithm, namely second-order differentiation combined with a simulated annealing particle swarm optimization algorithm based on sine adaptive weights (DWSA-PSO), was proposed for the separation of overlapping IMS peaks formed by glycan isomers. DWSA-PSO first performed second-order differentiation to automatically determine the number of components in overlapping peaks and exclude impossible single-peak combinations. It then introduced sinusoidal adaptive weights and a simulated annealing mechanism to improve the algorithm's search capability and global optimization performance, thereby enabling accurate and efficient separation of individual peaks. To evaluate the performance of DWSA-PSO and its application to the separation of glycan isomers, multiple sets of overlapping peaks with different degrees of overlap were simulated, and various types of multi-component overlapping peaks were formed using six disaccharide and four trisaccharide isomers. The experimental results consistently demonstrated that the DWSA-PSO algorithm outperformed both the improved particle swarm optimization (IPSO) algorithm and the dynamic inertia weight particle swarm optimization (DIWPSO) algorithm in terms of separation accuracy, running time, and fitness values. In addition, the DWSA-PSO algorithm was successfully applied to the separation of glycan isomers in malt milk beverage. All these results reveal the capability of the DWSA-PSO algorithm to facilitate the accurate identification of glycan isomers. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO), as a critical transcellular messenger, participates in a variety of physiological and pathological processes. However, its real-time detection still faces challenges due to its short half-life and trace amounts. Here, MWCNTs@COF-366-Co was prepared by in situ growth of a cobalt porphyrin-based covalent organic framework (COF-366-Co) on multi-walled carbon nanotubes (MWCNTs), and a unique biosensing platform for ultrasensitive real-time NO determination was established. Remarkably, MWCNTs@COF-366-Co contains plenty of atomically arranged M–N4 active sites for electrocatalysis, which provides more efficient electron transfer pathways and resolves the random arrangement issue of active sites. COF-366-Co with a high surface area contains a large number of exposed active M–N4 sites, providing faster NO transport/diffusion and more efficient electron transfer pathways. Due to the synergy of atomic-level periodic structural features of COF-366-Co and high conductivity of MWCNTs, the MWCNTs@COF-366-Co electrochemical biosensor exhibited excellent NO determination performance in a wide range from 0.09 to 400 μM, with high sensitivity (8.9 μA μM−1 cm−2) and a low limit of detection (16 nM). Moreover, the biosensor has been successfully used to sensitively monitor NO molecules released from human umbilical vein endothelial cells (HUVECs). This research not only designed a multifunctional intelligent biosensor platform, but also provided a broad prospect for continuous dynamic monitoring of the activity of living cells and their released metabolites. [ABSTRACT FROM AUTHOR]

Protein glycosylation is of great significance in various physiological processes. Nevertheless, it remains a huge challenge to identify glycopeptides in complex biosamples by the direct mass spectrometry analysis due to the low ion efficiency and low abundance of glycopeptides. In this study, a novel hydrogel (denoted as ZIF-8/SAP) with a stable three-dimensional (3D) network structure and excellent hydrophilicity was successfully fabricated to capture glycopeptides with high efficiency. Owing to the unique characteristics, ZIF-8/SAP exhibited great selectivity (1 : 1000), great sensitivity (1 fmol μL−1), large binding capacity (300 mg g−1) and satisfactory reusability (seven cycles). Inspired by the great enrichment performance of the as-prepared material toward glycopeptides, ZIF-8/SAP was further applied to capture glycopeptides from a real human serum sample. The experimental results demonstrated that 217 N-glycosylation sites were identified in 283 N-glycopeptides, corresponding to 95 glycoproteins identified from 10 μL human serum by the nano-LC-MS/MS analysis, revealing the great potential of the novel ZIF-8/SAP hydrogel for glycopeptide enrichment and glycoproteomic research. [ABSTRACT FROM AUTHOR]