基于双向电泳-MALDI质谱分析的蛋白组学本科教学实验

蛋白质组学已成为研究生物学过程及直接描述细胞和组织变化的有力手段。双向电泳结合生物质谱技术是蛋白质组学研究中的最常用的蛋白质分离和鉴定技术之一。该项技术是生物技术专业本科生必须掌握的一门实验技术。通过双向电泳-MALDI质谱分析的蛋白组学本科教学实验的设计,建立了完善的本科教学实验方案,让大型仪器全面支撑本科教学实验,进一步提高了大型仪器的利用率。     

Clathrate nanostructures for mass spectrometry

The ability of mass spectrometry to generate intact biomolecular ions efficiently in the gas phase has led to its widespread application in metabolomics, proteomics, biological imaging, biomarker discovery and clinical assays (namely neonatal screens). Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization have been at the forefront of these developments. However, matrix application complicates the use of MALDI for cellular, tissue, biofluid and microarray analysis and can limit the spatial resolution because of the matrix crystal size (typically more than 10 mum), sensitivity and detection of small compounds (less than 500 Da). Secondary-ion mass spectrometry has extremely high lateral resolution (100 nm) and has found biological applications although the energetic desorption/ionization is a limitation owing to molecular fragmentation. Here we introduce nanostructure-initiator mass spectrometry (NIMS), a tool for spatially defined mass analysis. NIMS uses 'initiator' molecules trapped in nanostructured surfaces or 'clathrates' to release and ionize intact molecules adsorbed on the surface. This surface responds to both ion and laser irradiation. The lateral resolution (ion-NIMS about 150 nm), sensitivity, matrix-free and reduced fragmentation of NIMS allows direct characterization of peptide microarrays, direct mass analysis of single cells, tissue imaging, and direct characterization of blood and urine.     

水产品中环境雌激素特性及检测技术研究进展

环境雌激素会干扰生物体内雌激素的正常分泌,使生物体的生殖功能或免疫功能出现异常。环境雌激素可以分为合成雌激素、生物源雌激素和环境化学污染物,检测方法主要有气相色谱（GC）法、高效液相色谱（HPLC）法、气相色谱-质谱联用（GC-MS）法、化学发光免疫分析（CLIA）法、分子印迹（MIT）法。目前,我国对环境雌激素的研究...     

Unidirectional rotary motion in a molecular system.

The conversion of energy into controlled motion plays an important role in both man-made devices and biological systems. The principles of operation of conventional motors are well established, but the molecular processes used by 'biological motors' such as muscle fibres, flagella and cilia to convert chemical energy into co-ordinated movement remain poorly understood. Although 'brownian ratchets' are known to permit thermally activated motion in one direction only, the concept of channelling random thermal energy into controlled motion has not yet been extended to the molecular level. Here we describe a molecule that uses chemical energy to activate and bias a thermally induced isomerization reaction, and thereby achieve unidirectional intramolecular rotary motion. The motion consists of a 120 degrees rotation around a single bond connecting a three-bladed subunit to the bulky remainder of the molecule, and unidirectional motion is achieved by reversibly introducing a tether between the two units to energetically favour one of the two possible rotation directions. Although our system does not achieve continuous and fast rotation, the design principles that we have used may prove relevant for a better understanding of biological and synthetic molecular motors producing unidirectional rotary motion.     

Quantitative dynamics and binding studies of the 20S proteasome by NMR

The machinery used by the cell to perform essential biological processes is made up of large molecular assemblies. One such complex, the proteasome, is the central molecular machine for removal of damaged and misfolded proteins from the cell. Here we show that for the 670-kilodalton 20S proteasome core particle it is possible to overcome the molecular weight limitations that have traditionally hampered quantitative nuclear magnetic resonance (NMR) spectroscopy studies of such large systems. This is achieved by using an isotope labelling scheme where isoleucine, leucine and valine methyls are protonated in an otherwise highly deuterated background in concert with experiments that preserve the lifetimes of the resulting NMR signals. The methodology has been applied to the 20S core particle to reveal functionally important motions and interactions by recording spectra on complexes with molecular weights of up to a megadalton. Our results establish that NMR spectroscopy can provide detailed insight into supra-molecular structures over an order of magnitude larger than those routinely studied using methodology that is generally applicable.     

家蚕血液肽聚糖识别蛋白的鉴定和生物信息学分析

采用双向电泳和质谱技术分析、鉴定了家蚕血液中存在的肽聚糖识别蛋白(PGRP).为了进一步研究该蛋白的结构和功能,采用了生物信息学方法对其理化性质、疏水性/亲水性、信号肽、二级结构、三级结构、亚细胞定位和GO功能注释等进行了分析.结果发现,肽聚糖识别蛋白是一种分泌类蛋白,可能具有信号肽.其主要分子功能为肽聚糖受体,在生物学进程中发挥的主要作用为肽聚糖代谢和先天性免疫应答.     

Chiral recognition in dimerization of adsorbed cysteine observed by scanning tunnelling microscopy

Stereochemistry plays a central role in controlling molecular recognition and interaction: the chemical and biological properties of molecules depend not only on the nature of their constituent atoms but also on how these atoms are positioned in space. Chiral specificity is consequently fundamental in chemical biology and pharmacology and has accordingly been widely studied. Advances in scanning probe microscopies now make it possible to probe chiral phenomena at surfaces at the molecular level. These methods have been used to determine the chirality of adsorbed molecules, and to provide direct evidence for chiral discrimination in molecular interactions and the spontaneous resolution of adsorbates into extended enantiomerically pure overlayers. Here we report scanning tunnelling microscopy studies of cysteine adsorbed to a (110) gold surface, which show that molecular pairs formed from a racemic mixture of this naturally occurring amino acid are exclusively homochiral, and that their binding to the gold surface is associated with local surface restructuring. Density-functional theory calculations indicate that the chiral specificity of the dimer formation process is driven by the optimization of three bonds on each cysteine molecule. These findings thus provide a clear molecular-level illustration of the well known three-point contact model for chiral recognition in a simple bimolecular system.     

Cell membranes impermeable to NH3

Classically, there is a direct correlation between the lipophilic nature of a molecule and its rate of permeation across a biological membrane, so cell membranes should be more permeable to small, neutral molecules than they are to charged molecular species of similar size. Consequently, the distribution of NH+4 in biological systems is generally believed to be due to the rapid diffusion and equilibration of lipophilic NH3 across cell membranes and the accumulation of NH+4 to be governed by pH differences between compartments. Here we report that renal tubule cells from the medullary thick ascending limb of Henle have an apical membrane which is not only virtually impermeable to NH3, but is also highly permeable to NH+4. These remarkable properties have been incorporated into a model which explains how this renal epithelium can mediate vectorial movement of NH+4 between compartments of equal pH.     

1,1-二苯基乙烯存在下甲基丙烯酸甲酯的自由基聚合研究

以偶氮二异丁腈（AIBN）为引发剂,1,1-二苯基乙烯（DPE）为添加剂,对甲基丙烯酸甲酯（MMA）的自由基聚合过程进行了研究,并通过GPC、1H-NMR、ESI-MS表征了PMMA大分子的结构。结果表明,在DPE存在下,体系在一定程度上具备可控聚合的特征：聚合物分子量随单体转化率增加而增加,分子量分布较窄;DPE用量越大,相同时间下单体转化率越低;相同单体转化率下,DPE用量越大,聚合物的相对分子质量越低。PMMA大分子主要结构为一个DPE端基的链自由基和一个PMMA链自由基间偶合终止生成的含有一个DPE单元的特征结构。     

Computational design of receptor and sensor proteins with novel functions

The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.     

Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin

Fibroblast growth factors (FGFs) are a large family of structurally related proteins with a wide range of physiological and pathological activities. Signal transduction requires association of FGF with its receptor tyrosine kinase (FGFR) and heparan sulphate proteoglycan in a specific complex on the cell surface. Direct involvement of the heparan sulphate glycosaminoglycan polysaccharide in the molecular association between FGF and its receptor is essential for biological activity. Although crystal structures of binary complexes of FGF-heparin and FGF-FGFR have been described, the molecular architecture of the FGF signalling complex has not been elucidated. Here we report the crystal structure of the FGFR2 ectodomain in a dimeric form that is induced by simultaneous binding to FGF1 and a heparin decasaccharide. The complex is assembled around a central heparin molecule linking two FGF1 ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF1 molecules but only one receptor chain. The structure of the FGF1-FGFR2-heparin ternary complex provides a structural basis for the essential role of heparan sulphate in FGF signalling.     

N3^—杀虫效应的确定

本从Ｎ×－３的结构，电子组态，价键出发来说明它所具有的高度活泼性和生物学效庆，交用酶学试验和杀虫效果来证实；对Ｎ＾－３的杀虫机制，特点进行了探讨；对Ｎ＾－３的杀虫应用前景也作了估计。     

海带硫酸多糖降解研究进展

海带硫酸多糖是一种高分子量杂多糖，具有多种生物学功能．介绍了降解海带硫酸多糖的有关方法及其功能．根据近几年国内外相关文献以及本实验室的工作，海带硫酸多糖的降解可分为酸解法、碱解法、酶解法、盐解法、氧化降解以及超声波、γ-射线、自由基降解法．自由基降解海带硫酸多糖是一种可行的可用于大量制备低分子量多糖的新技术．降解之后的海带硫酸多糖有广泛的生物学功能，具有巨大的开发前景，这种方法也可用于其它多糖的降解．     

A molecular information ratchet

Motor proteins and other biological machines are highly efficient at converting energy into directed motion and driving chemical systems away from thermodynamic equilibrium. But even though these biological structures have inspired the design of many molecules that mimic aspects of their behaviour, artificial nanomachine systems operate almost exclusively by moving towards thermodynamic equilibrium, not away from it. Here we show that information about the location of a macrocycle in a rotaxane-a molecular ring threaded onto a molecular axle-can be used, on the input of light energy, to alter the kinetics of the shuttling of the macrocycle between two compartments on the axle. For an ensemble of such molecular machines, the macrocycle distribution is directionally driven away from its equilibrium value without ever changing the relative binding affinities of the ring for the different parts of the axle. The selective transport of particles between two compartments by brownian motion in this way bears similarities to the hypothetical task performed without an energy input by a 'demon' in Maxwell's famous thought experiment. Our observations demonstrate that synthetic molecular machines can operate by an information ratchet mechanism, in which knowledge of a particle's position is used to control its transport away from equilibrium.     

Cloning and sequence analysis of cDNAs encoding two distinct somatostatin precursors found in the endocrine pancreas of anglerfish

Complementary DNAs for two distinct anglerfish somatostatin peptides (termed I and II) have been cloned in bacterial plasmids and sequenced. The nucleotide sequence for somatostatin I encodes a large percursor peptide (molecular weight 13,300) in which the somatostatin hormones is at the carboxyl terminus. The predicted 14-amino acid sequence for anglerfish somatostatin I is the same as mammalian somatostatin. Somatostatin II is also synthesized as part of a larger precursor (molecular weight 14,100) with the presumptive somatostatin hormone also at the carboxyl terminus. The 14-amino acid sequence of somatostatin II differs from somatostatin I at two internal residues (Tyr in place of Phe 7 and Gly in place of Thr 10). The two different somatostatins may have distinct biological activities. Homologies in the amino acid sequences of the two peptides outside the somatostatin moiety suggest other regions of the molecules have biological functions.     

Proteome survey reveals modularity of the yeast cell machinery

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.     

Epstein--Barr virus-induced cell fusion

Serological and molecular biological studies have shown an association between Epstein--Barr virus (EBV) and nasopharyngeal carcinoma. Although it has been shown that the epithelioid tumour cells carry EBV genomes, they are apparently devoid of receptors for EBV (H.W., unpublished observations). Other have suggested that fusion of EBV carrying cells with epithelial cells may be the mode of entry of the virus into cells unable to absorb the virus and that this may be mediated by one of the known syncytium-forming viruses which inhabit the respiratory tract (for example, members of the paramyxovirus group). de Thé and colleagues suggested that intercellular bridges could be seen in NPC tumour material. We have developed a technique which permits the preparation of stable monolayers of viable human lymphoblastoid cell lines. Using this technique we have now demonstrated that EBV can induce fusion between EBV-superinfected lymphoblastoid cells and cells devoid of EBV receptors.     

癌症相关促凋亡蛋白Par-4的信号转导途径预测研究

利用支持向量机(SVM)技术构建Par-4关联的蛋白质相互作用网络,预测出与Par-4有相互作用的蛋白质82个;这些蛋白质按照功能划分为8大类,主要包括:蛋白激酶、泛素化蛋白酶、死亡受体相关因子、与细胞周期或DNA复制相关蛋白质、调节蛋白质、与疾病相关蛋白质、具有特定结构域结合蛋白质和其他蛋白质等。结合文献挖掘和数据库检索信息,推断出了Par-4的2条可能新的信号转导途径。首次预测到Par-4与一大类泛素化蛋白有密切的关系。研究发现,Par-4与多种蛋白质具有复杂的相互作用,并且,在多个细胞凋亡途径中扮演了重要角色。     

人体血液的光声谱

本文对人体全血和血液中的各个成分以及某些病人的血液进行了光声谱的测量.实验结果表明:全血的光声谱主要反映红细胞内血红蛋白的光吸收特性,其特征取决于血红蛋白中血红素的分子结构。血红蛋白浓度改变和血红素分子结构不同,均使血红蛋白光声谱发生变化。因此,光声谱技术为研究血红蛋白提供了一种新的方法.     

固相合成MAPO-5分子筛及其在甲苯催化燃烧中的应用

以二正丙胺磷酸盐(DPA·H₃PO₄)和四乙基溴化铵(TEABr)为双模板剂,采用简单、绿色、高效的固相研磨法合成了多级孔杂原子磷酸铝分子筛CeAPO-5和CoAPO-5;为提升催化性能,增加分子筛中铈离子的含量,制备了一系列不同铈掺杂量的CeAPO-5分子筛,包括CeAPO-5分子筛(w(Ce)=2%)、1.2CeAPO-5分子筛(w(Ce)=2.4%)和2CeAPO-5分子筛(w(Ce)=4%)。通过X-射线衍射(XRD)、扫描电子显微镜(SEM)、N₂吸脱附测试(BET)、固体紫外可见(UV-Vis)漫反射光谱和X射线光电子能谱(XPS)等手段对所合成分子筛进行表征,同时以甲苯为目标污染物,考察了分子筛催化剂在催化燃烧反应中的催化性能。结果表明,所合成的分子筛都具有典型的AFI结构,结晶度较高,且Ce离子和Co离子被成功引入到分子筛骨架中;CeAPO-5分子筛在甲苯催化燃烧反应中较CoAPO-5分子筛具有更好的催化性能;在将金属Ce的含量从2%提高到4%的过程中,催化剂催化氧化甲苯的活性也有所提高,当反应温度为400 ℃时,2CeAPO-5分子筛的甲苯转化率接近100%;通过对催化剂在360 ℃时催化氧化甲苯的稳定性进行研究发现,2CeAPO-5分子筛在经过30 h的反应后仍保持70%左右的甲苯转化率。