Metal organic layers enabled cell surface engineering coupling biomembrane fusion for dynamic membrane proteome profiling.

Systematically dissecting the highly dynamic and tightly communicating membrane proteome of living cells is essential for the system-level understanding of fundamental cellular processes and intricate relationship between membrane-bound organelles constructed through membrane traffic. While extensive efforts have been made to enrich membrane proteins, their comprehensive analysis with high selectivity and deep coverage remains a challenge, especially at the living cell state. To address this problem, we developed the cell surface engineering coupling biomembrane fusion method to map the whole membrane proteome from the plasma membrane to various organelle membranes taking advantage of the exquisite interaction between two-dimensional metal-organic layers and phospholipid bilayers on the membrane. This approach, which bypassed conventional biochemical fractionation and ultracentrifugation, facilitated the enrichment of membrane proteins in their native phospholipid bilayer environment, helping to map the membrane proteome with a specificity of 77% and realizing the deep coverage of the HeLa membrane proteome (5087 membrane proteins). Furthermore, membrane N-phosphoproteome was profiled by integrating the N-phosphoproteome analysis strategy, and the dynamic membrane proteome during apoptosis was deciphered in combination with quantitative proteomics. The features of membrane protein N-phosphorylation modifications and many differential proteins during apoptosis associated with mitochondrial dynamics and ER homeostasis were found. The method provided a simple and robust strategy for efficient analysis of membrane proteome, offered a reliable platform for research on membrane-related cell dynamic events and expanded the application of metal-organic layers.

A photo-oxidation driven proximity labeling strategy enables profiling of mitochondrial proteome dynamics in living cells.

Mapping the proteomic landscape of mitochondria with spatiotemporal precision plays a pivotal role in elucidating the delicate biological functions and complex relationship with other organelles in a variety of dynamic physiological processes which necessitates efficient and controllable chemical tools. We herein report a photo-oxidation driven proximity labeling strategy to profile the mitochondrial proteome by light dependence in living cells with high spatiotemporal resolution. Taking advantage of organelle-localizable organic photoactivated probes generating reactive species and nucleophilic substrates for proximal protein oxidation and trapping, mitochondrial proteins were selectively labeled by spatially limited reactions in their native environment. Integration of photo-oxidation driven proximity labeling and quantitative proteomics facilitated the plotting of the mitochondrial proteome in which up to 310 mitochondrial proteins were identified with a specificity of 64% in HeLa cells. Furthermore, mitochondrial proteome dynamics was deciphered in drug resistant Huh7 and LPS stimulated HMC3 cells which were hard-to-transfect. A number of differential proteins were quantified which were intimately linked to critical processes and provided insights into the related molecular mechanisms of drug resistance and neuroinflammation in the perspective of mitochondria. The photo-oxidation driven proximity labeling strategy offers solid technical support to a highly precise proteomic platform in time and finer space for more knowledge of subcellular biology.

Simultaneous enrichment and sequential separation of glycopeptides and phosphopeptides with poly-histidine functionalized microspheres.

Protein phosphorylation and glycosylation coordinately regulate numerous complex biological processes. However, the main methods to simultaneously enrich them are based on the coordination interactions or Lewis acid-base interactions, which suffer from low coverage of target molecules due to strong intermolecular interactions. Here, we constructed a poly-histidine modified silica (SiO2@Poly-His) microspheres-based method for the simultaneous enrichment, sequential elution and analysis of phosphopeptides and glycopeptides. The SiO2@Poly-His microspheres driven by hydrophilic interactions and multiple hydrogen bonding interactions exhibited high selectivity and coverage for simultaneous enrichment of phosphopeptides and glycopeptides from 1,000 molar folds of bovine serum albumin interference. Furthermore, "on-line deglycosylation" strategy allows sequential elution of phosphopeptides and glycopeptides, protecting phosphopeptides from hydrolysis during deglycosylation and improving the coverage of phosphopeptides. The application of our established method to HT29 cell lysates resulted in a total of 1,601 identified glycopeptides and 694 identified phosphopeptides, which were 1.2-fold and 1.5-fold higher than those obtained from the co-elution strategy, respectively. The SiO2@Poly-His based simultaneous enrichment and sequential separation strategy might have great potential in co-analysis of PTMs-proteomics of biological and clinic samples.

Oleic acid as a protein ligand improving intestinal absorption and ocular benefit of fucoxanthin in water through protein-based encapsulation.

In this work, fucoxanthin-oleic acid-protein complexes were constructed to improve the dispersibility and intestinal absorption of fucoxanthin in water. The in vivo absorption/antioxidant capacity was evaluated using a mouse model, and the binding processes were investigated using multi-spectroscopic methods and molecular docking. Results showed that the oleic acid-protein delivery system dramatically improved the absorption of fucoxanthin mainly in its original form. When the molar ratio of oleic acid to bovine serum albumin (BSA) was 4 : 1, the plasma response level of fucoxanthin at 4 h could reach 91.25% that of the pure soybean oil delivery system (336.9 pg mL-1vs. 369.2 pmol mL-1). Furthermore, the loading capacity of BSA to fucoxanthin was increased 5 times when oleic acid acted as a protein ligand. Fucoxanthin, oleic acid and BSA can form complexes with good water dispersibility (transmittance nearly 90% and particle size 265 nm) at the molar ratio of 5 : 4 : 1. Spectral analysis and molecular docking indicated that oleic acid and fucoxanthin have different binding domains in BSA and that fucoxanthin can bind to the hydrophobic cavity of BSA in a static manner. After administration of fucoxanthin-oleic acid-BSA complexes for 15 days in mice, only fucoxanthinol accumulation was discovered in eyes and the ocular antioxidant capability increased by 71.02%. These results suggest that the oleic acid-protein delivery system may be useful in facilitating the application of fat-soluble active substances to hydrophilic food systems.