N-糖组富集方法研究进展

糖基化是一种重要的蛋白质翻译后修饰,使蛋白质具有更多样的功能.N-糖基化作为蛋白质主要的糖基化类型之一,与许多生命活动密切相关,在疾病发生发展中起关键作用.因此,研究糖蛋白N-糖链具有重要意义.生物质谱技术是糖组学研究的重要工具之一,然而,由于糖链结构复杂、丰度低及质谱离子化效率低等原因,基于质谱的N-糖组学研究还面临...     

构筑“甜蜜的”纳米结构促进细胞内吞

复旦大学陈国颂教授等通过巧妙的高分子链结构设计,共价键与非共价相互作用的分级协同,精确地控制了糖功能基元在纳米粒子表面的分布,研究了糖功能基元分布对细胞内吞的影响.     

糖芯片的研究进展

糖芯片是生物芯片的一种,如基因芯片对于基因研究和蛋白质芯片对于蛋白质组研究一样,糖芯片在糖组学的研究中同样也将扮演重要的角色。本文系统介绍了糖芯片的制备流程及其应用,以及在糖芯片研发开发中的技术障碍。     

非共价键法改性碳纳米管的研究进展

阐述了非共价键改性在碳纳米管功能化方面的应用进展,详细介绍了表面活性剂修饰、小分子的π-π堆积相互作用、聚合物的缠绕和包覆、生物大分子的包裹和吸附、内嵌填充修饰等改性方式的研究现状,并提出了非共价键法修饰碳纳米管未来改进的方向,探讨了不同修饰物与碳纳米管之间的相互作用机制。其中,含有共轭基团或芳基基团的聚合物,可以通过其共轭或芳基基团与碳纳米管间的π-π相互作用和范德华作用,实现对碳纳米管的非共价物理包覆。经聚合物功能化的碳纳米管在电池、催化剂、生物传感器和电化学装置上有较好的应用前景。此外,生物大分子对碳纳米管的非共价修饰不仅可以改善其在生物体系中的水溶性,而且通过合理设计还可以避免蛋白质、核酸等生物分子的非特异性吸附,从而得到具有特异性的生物分子-碳纳米管复合体系。     

糖-蛋白质特异性识别在生物材料领域的应用

糖类不仅是组成生命体的基本物质之一,还是很多生理和病理过程中分子识别和细胞间相互作用的基础。糖的生物学功能可通过与蛋白质的相互作用来实现。这种可逆的特异性识别在信号传导、细胞粘附、增殖、分化、病菌感染和免疫应答等生命过程中具有重要意义。本文着重介绍了糖-蛋白质相互作用的种类和机理,综述了糖-蛋白质特异性识别作用在生物材料领域的应用进展,包括将糖类作为靶向分子修饰到纳米粒子或载体分子表面进行药物传递和基因转染,利用糖-蛋白质非共价作用力进行分子组装,以及制备糖基化表面用于调控蛋白粘附和细胞行为。由于糖类特殊的生物学性能和种类的多样性,基于糖-蛋白质特异性识别有望用于制备更加实用和智能的生物材料。     

基于质谱的糖蛋白/糖链定量技术研究进展

糖基化修饰是一种重要的蛋白质翻译后修饰。糖基化修饰的蛋白质在生命体内具有重要的生物学功能。研究糖蛋白含量以及蛋白上糖链变化对于阐明糖基化修饰的功能具有重要的意义,也是当今的研究热点。本文就糖蛋白和糖链定量方法的研究进展和应用做了简要概述。     

DNA修饰电极的研究(ⅠΧ)——DNA探针在金基底上的固定、表征及其表面分子杂交

采用巯基化合物自组装 /共价键合反应的逐层固定方法将双链 DNA固定到金表面得到 DNA修饰电极 ,并对该电极表面进行了电化学和 X射线光电子能谱表征 .研究了电极表面固定化 DNA的表面分子杂交 .对开发电化学基因诊断芯片和基因传感器具有一定意义     

基于ConA富集和LTQ-FT质谱鉴定的小鼠肝脏内质网N-糖蛋白质组研究

糖基化修饰是生物体内复杂和重要的蛋白质翻译后修饰方式之一.N-糖基化蛋白质在内质网中进行合成的过程中,所有的N-糖链都以甘露糖和葡萄糖结尾,而凝集素ConA对以甘露糖结尾的糖链有较高的亲和性,可以用来富集在内质网中合成的N-糖蛋白质.本文据此提出了一种基于内质网分离和凝集素ConA富集的复杂样品N-糖基化位点研究策略.通过使用高准确度的质谱线性离子阱-傅立叶变换回旋离子共振质谱对N-糖蛋白质进行鉴定,并对N-糖基化位点进行确定.我们采用模式生物C57BL/6J肝脏作为生物样本,在生物水平和质谱水平分别进行了3次重复,共鉴定了212个N-糖蛋白质的323个N-糖基化位点.在这些蛋白中,131个是Swissprot库中已确认的N-糖蛋白质.此方法富集的糖蛋白,糖型统一,有利于样品的分离和PNGaseF酶切作用,提高了鉴定的效率.对鉴定的212个N-糖蛋白质的定位和功能进行了分析,本文鉴定的N-糖蛋白质对现有的鼠肝N-糖蛋白质数据库进行了有效的补充.     

表面等离子体子共振技术实时研究蛋白质在修饰表面的静电吸附行为

研究蛋白质在固相表面的静电吸附特性,进而控制蛋白质在修饰表面的静电吸附尤为重要,表面等离子体子共振可以检测金属表面吸附物质厚度和折射率的变化^[1]。这种技术已在研究生物分子相互作用^[2]和考察自组装单层的形成^[3]及蛋白质在固体表面吸附行为^[9-11]等方面得到广泛的应用。对蛋白质在固体表面吸附行为的研究多为考察不同的蛋白质在不同的修饰表面的吸附行为。然而,对蛋白质在修饰表面静电吸附的本质影响因素的研究却少有报道^[4]。本文使用表面等离子体子共振技术实时研究了蛋白质在甲羧基化葡聚糖修饰表面的静电吸附与溶液pH值及离子强度的依赖关系。     

硅基芯片表面化学性质对蛋白质固定化的影响

制备蛋白质芯片的关键在于将蛋白质固定到芯片表面并保持其生物学活性.本实验中,我们分别采用物理吸附、直接化学固定、加入间隔臂化学固定和生物亲和作用固定的方法将癌胚抗原(CEA)抗体固定到硅基芯片的二氧化硅表面.基于抗原-抗体的特异性相互作用,利用双抗体夹心酶联免疫法(ELISA)评价各种方法固定抗体的效果.实验结果表明,在修饰有氨基的表面采用戊二醛作为偶联试剂固定CEA抗体具有最高的偶联效率,引入多聚赖氨酸(poly-L-lysine)作为间隔臂可以显著增强固定效果,并可进一步降低非特异性吸附.而利用生物亲和作用固定CEA抗体也可获得较好的固定效果,但是非特异性吸附较严重.     

Glyco-macroligand microarray with controlled orientation and glycan density

We report a new type of glycan microarray, namely, oriented and density-controlled glyco-macroligand microarray based on end-point immobilization of glycopolymer that was accompanied with boronic acid (BA) ligands in different sizes as detachable "temporary molecular spacers". Briefly, an O-cyanate chain-end functionalized lactose-containing glycopolymer was pre-complexed with polyacrylamide-BA, lysozyme-BA, and bovine serum albumin (BSA)-BA conjugates as macromolecular spacers first and then immobilized onto an amine-functionalized glass slide via isourea bond formation both at pH 10.3, respectively. Subsequently, the macromolecular spacers were detached from the immobilized glycopolymers at pH 7.4 so as to afford the oriented and density controlled glycopolymer microarrays. The spaced glycopolymer microarray showed enhanced lectin (Arachis hypogaea) binding compared to a non-spaced one. Among them, the polyacrylamide-BA spaced glycopolymer showed the highest level of lectin binding compared to lysozyme-BA- and BSA-BA-spaced glycopolymers. Furthermore, SPR results confirmed the same trend of density-dependent lectin binding as the glycoarray. This glyco-macroligand microarray platform permits variations of glycan density in the polymer, glycopolymer density and its orientation on the microarray surface and thus will provide a versatile tool for profiling glycan recognition for both basic biological research and practical applications.     

Immobilization of sunflower trypsin inhibitor (SFTI-1) peptide onto a gold surface and analysis of its interaction with trypsin

Sunflower trypsin inhibitor (SFTI-1) derived peptide having one disulfide bond could be immobilized via a thiol-disulfide exchange reaction onto a gold surface on a quartz crystal microbalance (QCM) chip. This permitted quantitative analysis of the specific interaction with trypsin.     

Novel well‐defined glycopolymers synthesized via the reversible addition fragmentation chain transfer process in aqueous media

We describe here the direct synthesis of novel gluconamidoalkyl methacrylamides by reacting D ‐gluconolactone with aminoalkyl methacrylamides. The glycomonomers were then successfully polymerized via the reversible addition‐fragmentation chain transfer process (RAFT) using 4‐cyanopentanoic acid dithiobenzoate (CTP) as chain transfer agent and 4,4′‐azobis(4‐cyanovaleric acid) (ACVA) as the initiator in aqueous media. Well‐defined polymers were obtained as revealed by gel permeation chromatography. Diblock copolymers were then synthesized by the macro‐CTA approach. The cationic glycopolymers were subsequently used in the formation of nanostructures via the complexation with plasmid DNA. As noted by dynamic light scattering, monodisperse nanoparticles were obtained via the electrostatic interaction of the cationic glycopolymer with DNA. The sizes of the nanoparticles formed were found to be stable and independent of pH. In vitro cell viability studies of the glycopolymers were carried out using HELA cell lines. The RAFT synthesized glycopolymers and cationic glyco‐copolymers revealed to be nontoxic. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 614–627, 2009     

One‐Pot Multicomponent Synthesis of Glycopolymers through a Combination of Host–Guest Interaction,Thiol‐ene,and Copper‐Catalyzed Click Reaction in Water

There is a common phenomenon that the heterogeneity of natural oligosaccharides contains various sugar units, which can be used to enhance affinity and selectivity toward a specific receptor, so the synthesis of heterogeneous glycopolymers is always an important issue in the glycopolymer field. Herein, this study conducts a one‐pot method to prepare polyrotaxane‐based heteroglycopolymers anchored with different sugar units and fluorescent moieties via the combination of host–guest interaction, thiol‐ene, and copper‐catalyzed click chemistry in water. Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, gel permeation chromatography, X‐ray diffraction, and Ellman's assay test are used in the paper to characterize the compounds. Quartz crystal microbalance‐dissipation (QCD‐D) experiments and bacterial adhesion assay are utilized to study the interactions of polyrotaxane‐based heteroglycopolymers with Con A and Escherichia coli . The results reveal that polyrotaxanes (PRs) with mannose and glucose present better specificity toward Con A and E. coli than PRs with glucose due to synergistic effects.     

Thermal stability and biodegradation of novel D-mannose based glycopolymers

This paper presents the thermal stability and biodegradability testing of new glycopolymers obtained by copolymerization of a novel D-mannose based oligomer with 2-hydroxypropyl acrylate and 2-hydroxypropyl methacrylate. The thermal analysis of these glycopolymers was investigated by thermogravimetry and the glass transition temperature was determined by DSC. While the acrylate derived glycopolymer has values of the glass transition temperature below 0 °C, the methacrylate derivative has positive values, above 50 °C. The biodegradation studies of the glycopolymers were carried out in a liquid medium, using pure cultures of two microorganisms, Zymomonasmobilis and Trichodermareesei. The weight losses of the new plastic materials were significant (almost 40%) and the best results were assessed for the 2-hydroxypropyl acrylate glycopolymer in the presence of both Z. mobilis and T. reesei. Microscopy showed that both microorganisms were present on the surface of the new glycopolymers and developed small colonies while modifying their surface. The changes inside the morphology of the polymeric materials structure were drastic and were studied via SEM analysis.     

Surface Modification Based on Diselenide Dynamic Chemistry: Towards Liquid Motion and Surface Bioconjugation

Surface modification is an important technique in fields, such as, self‐cleaning, surface patterning, sensing, and detection. The diselenide bond was shown to be a dynamic covalent bond that can undergo a diselenide metathesis reaction simply under visible light irradiation. Herein we develop this diselenide dynamic chemistry into a versatile surface modification method with a fast response and reversibility. The diselenide bond could be modified onto various substrates, such as, PDMS, quartz, and ITO conductive film glass. Different functional diselenide molecules could then be immobilized onto the surface via diselenide metathesis reaction. We demonstrated that by using this modification method we could achieve liquid motion in a capillary tube under light illumination. We also show that this approach has the potential to serve as an efficient modification method for surface bioconjugation, which has practical applications in clinical usage.     

Glycopolymer Brushes for Specific Lectin Binding by Controlled Multivalent Presentation of N‐Acetyllactosamine Glycan Oligomers

A new multivalent glycopolymer platform for lectin recognition is introduced in this work by combining the controlled growth of glycopolymer brushes with highly specific glycosylation reactions. Glycopolymer brushes, synthetic polymers with pendant saccharides, are prepared by surface‐initiated atom transfer radical polymerization (SI‐ATRP) of 2‐O‐(N‐acetyl‐β‐d ‐glucosamine)ethyl methacrylate (GlcNAcEMA). Here, the fabrication of multivalent glycopolymers consisting of poly(GlcNAcEMA) is reported with additional biocatalytic elongation of the glycans directly on the silicon substrate by specific glycosylation using recombinant glycosyltransferases. The bioactivity of the surface‐grafted glycans is investigated by fluorescence‐linked lectin assay. Due to the multivalency of glycan ligands, the glycopolymer brushes show very selective, specific, and strong interactions with lectins. The multiarrays of the glycopolymer brushes have a large potential as a screening device to define optimal‐binding environments of specific lectins or as new simplified diagnostic tools for the detection of cancer‐related lectins in blood serum.

     

Sensitive electrogravimetric immunoassay of hepatitis B surface antigen through hyperbranched polymer bridge linked to multiple secondary antibodies

A method for using a hyperbranched polymer (HBP) as a bridge to link multiple secondary antibodies at HBP branches to amplify the detection response signal on a quartz crystal microbalance (QCM)-based sandwich-type immunosensor is reported. Carboxyl groups were prepared at multiple branches of HBP to make possible chemical binding between HBP and secondary antibodies via the carboxyl-amine reaction. The total mass of HBP and its linked multiple secondary antibodies were used to enhance the signal on a QCM chip in comparison with a simple sandwich-type immune reaction. By contrast, the proposed method could cause one antigen to analogously react with multiple secondary antibodies as a result of the branch structure of HBP. The strategy of using HBP as a bridge to link multiple secondary antibodies succeeded in quantitatively detecting the hepatitis B surface antigen (HBsAg). By employing demonstrated HBP bridge-linking, the frequency shift on a QCM chip was approximately 5 times greater than conventional methods without modification at secondary antibodies. The limit of detection of HBsAg was achieved as 2.0 ng mL^?1, lower than most of the values recorded in the literature measured by the QCM technique. Taking into account the general chemical interaction of immunoreaction, this method has the potential to amplify the signal in sensing many other analytes of interest.     

Protein Adsorption on a Glycosylated Polyacrylonitrile Surface: Monitoring with QCM and SPR

A simple and efficient method to fabricate a glycosylated surface on a polyacrylonitrile‐based film is described. Construction and protein adsorption processes were monitored in situ using a QCM. A PANCHEMA film was deposited on the gold surface of the quartz crystal, and the glycosylated surface was then constructed through surface modification. Con A and BSA were used as probes to study the specificity of this surface to proteins. It can recognize Con A, while showing no specific interaction with BSA. The binding affinity indicates the presence of strong multivalent interactions between Con A and the glucose residues (cluster glycoside effect). Reproducibility and repeatability of the glycosylated polymer surface are sufficient to allow specific adsorption of Con A.

     

Glyco-Modification of Protein With O-Cyanate Chain-End Functionalized Glycopolymer via Isourea Bond Formation

Glycoengineering aimed at the addition of carbohydrates to proteins is an attractive approach to alter the pharmacokinetic properties of proteins, such as enhancing stability and prolonging the duration of action. We report a novel protein glyco-modification of BSA and recombinant thrombomodulin with O-cyanate chain-end functionalized glycopolymer via isourea bond formation. The protein glycoconjugates were confirmed by SDS-PAGE, western blot, and MALDI-TOF mass spectrometry. Protein C activation activity of the glyco-modified recombinant thrombomodulin was confirmed, proving no interference with activity from the glycopolymer modification. The isourea bond formation under mild conditions was demonstrated as an alternative method for protein modification with polymers.