6 research outputs found
TurboID-EV: Proteomic Mapping of Recipient Cellular Proteins Proximal to Small Extracellular Vesicles
細胞外小胞の軌跡を照らす --細胞外小胞の標的細胞への取り込み機構の解明に貢献--. 京都大学プレスリリース. 2023-09-15.Extracellular vesicles (EVs), including exosomes, have been recognized as key mediators of intercellular communications through donor EV and recipient cell interaction. Until now, most studies have focused on the development of analytical tools to separate EVs and their applications for the molecular profiling of EV cargo. However, we lack a complete picture of the mechanism of EV uptake by the recipient cells. Here, we developed the TurboID-EV system with the engineered biotin ligase TurboID, tethered to the EV membrane, which allowed us to track the footprints of EVs during and after EV uptake by the proximity-dependent biotinylation of recipient cellular proteins. To analyze biotinylated recipient proteins from low amounts of input cells (corresponding to ∼10 μg of proteins), we developed an integrated proteomic workflow that combined stable isotope labeling with amino acids in cultured cells (SILAC), fluorescence-activated cell sorting, spintip-based streptavidin affinity purification, and mass spectrometry. Using this method, we successfully identified 456 biotinylated recipient proteins, including not only well-known proteins involved in endocytosis and macropinocytosis but also other membrane-associated proteins such as desmoplakin and junction plakoglobin. The TurboID-EV system should be readily applicable to various EV subtypes and recipient cell types, providing a promising tool to dissect the specificity of EV uptake mechanisms on a proteome-wide scale
液相分離におけるπ相互作用に関する研究
京都大学0048新制・課程博士博士(工学)甲第22701号工博第4748号新制||工||1742(附属図書館)京都大学大学院工学研究科材料化学専攻(主査)教授 大塚 浩二, 教授 松原 誠二郎, 教授 秋吉 一成学位規則第4条第1項該当Doctor of Philosophy (Engineering)Kyoto UniversityDFA
Moderate Molecular Recognitions on ZnO m-Plane and Their Selective Capture/Release of Bio-related Phosphoric Acids
Herein, we explore the hidden molecular recognition abilities of ZnO nanowires uniformly grown on the inner surface of an open tubular fused silica capillary via liquid chromatography. Chromatographic evaluation revealed that ZnO nanowires showed a stronger intermolecular interaction with phenylphosphoric acid than any other monosubstituted benzene. Furthermore, ZnO nanowires specifically recognized the phosphate groups present in nucleotides even in the aqueous mobile phase, and the intermolecular interaction increased with the number of phosphate groups. This discrimination of phosphate groups in nucleotides was unique to the rich (10[1 with combining macron]0) m-plane of ZnO nanowires with a moderate hydrophilicity and negative charge. The discrimination could be evidenced by the changes in the infrared bands of the phosphate groups on nucleotides on ZnO nanowires. Finally, as an application of the molecular recognition, nucleotides were separated by the number of phosphate groups, utilizing optimized gradient elution on ZnO nanowire column. Thus, the present results elucidate the unique and versatile molecular selectivity of well-known ZnO nanostructures for the capture and separation of biomolecules
Separation of saccharides using fullerene-bonded silica monolithic columns via π interactions in liquid chromatography
フラーレンを用いた糖鎖の分離手法を開発 --抗体等の糖タンパク質分離への応用に期待--. 京都大学プレスリリース. 2020-08-26.We report on a potential method to separate sugars by using the specific interaction between fullerenes and saccharides in liquid chromatography (LC). Aromatic rings with high electron density are believed to interact strongly with saccharides due to CH–π and/or OH–π interactions. In this study, the fullerene-bonded columns were used to separate saccharides by LC under aqueous conditions. As a result, 2-aminobenzamide-labeled glucose homopolymer (Glcs) was effectively separated by both C60 and C70 columns in the range of Glc-1 to Glc-20 and high blood glucose level being retained in greater quantity. Furthermore, similar separations were identified by LC–mass spectrometry with non-labeled glucose homopolymers. Theoretical study based on molecular dynamics and DFT calculation demonstrated that a supramolecular complex of saccharide–fullerene was formed through CH–π and/or OH–π interactions, and that the interactions between saccharide and fullerene increase with the increase units of the saccharide. Additionally, the C60 column retained disaccharides containing maltose, trehalose, and sucrose. In this case, it was assumed that the retention rates were determined by the difference of the dipole moment in each saccharide. These results suggest that the dipole-induced dipole interaction was dominant, and that maltose—with the higher dipole moment—was more strongly retained compared to other disaccharides having lower dipole moment
Separation of halogenated benzenes enabled by investigation of halogen–π interactions with carbon materials
The halogen–π (X–π) interaction is an intermolecular interaction between the electron-poor region of bonded halogen atoms and aromatic rings. We report an experimental evaluation of the halogen–π (X–π) interaction using liquid chromatography with carbon-material coated columns providing strong π interactions in the normal phase mode. A C₇₀-fullerene (C70)-coated column showed higher retentions for halogenated benzenes as the number of halogen substitutions increased as a result of X–π interactions. In addition, the strength of the X–π interaction increased in the order of F < Cl < Br < I. Changes to the UV absorption of C70 and the brominated benzenes suggested that the intermolecular interaction changed from the π–π interaction to X–π interaction as the number of bromo substitutions increased. Computer simulations also showed that the difference in dipole moments among structural isomers affected the strength of the π–π interaction. Furthermore, we concluded from small peak shifts in ¹H NMR and from computer simulations that the orbital interaction contributes to the X–π interactions. Finally, we succeeded in the one-pot separation of all isomers of brominated benzenes using the C70-coated column by optimizing the mobile phase conditions
Isotope Effects on Hydrogen Bonding and CH/CD−π Interaction
We study the isotope effect by liquid
chromatography (LC) with a variety of separation media under reversed
and normal phase conditions using the protiated and/or deuterated
compounds as the solutes. Results of reversed phase LC (RPLC) suggested
that the protiated compounds were more hydrophobic than that of deuterated
compounds due to the isotope effect based on the hydrogen bonding
between hydrogen atoms of isotopologues and hydroxy groups in the
mobile phase. The importance of the hydrogen bonding was also supported
by the separation of isotopologues with a silica stationary phase
on normal phase LC (NPLC), where the deuterated compounds showed stronger
hydrogen bonding to hydroxy groups on silanol. Additionally, we investigated
the difference of the strength between CH−π and CD−π
interactions. Comparison of free energies of isotopologues by RPLC
suggested that the CH−π interaction was slightly stronger
than CD−π interaction. Finally, we demonstrated the separation
of a few isotopologues on NPLC using a column coated with C<sub>70</sub>-fullerene, which is capable of strong π-based interactions,
by effective CH/CD−π interactions