人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱(Ti4+-IMAC)整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/L NaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti4+-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。     

纳豆菌细胞包埋材料的选择

以海藻酸钠（SA）和聚乙烯醇（PVA）为包埋材料，采用固定化细胞技术对纳豆菌生产纳豆激酶进行了研究。研究发现，在PVA中加入SA进行细胞包埋可获得渗透性能好强度高的固定化细胞，通过正交试验进一步确定：当PVA的浓度为11％，SA的浓度为1％，硼酸的浓度为5％，CaCl2的浓度为6％时，固定化细胞的强度最好，可反复使用6批次，活性也很高，产生的纳豆激酶酶活溶纤圈直径积达88mm^2／15μL。     

31P CP/MAS NMR of polycrystalline and immobilized phosphines and catalysts with fast sample spinning

Cross‐polarization (CP) at fast magic angle spinning (MAS) frequencies leads to a splitting of the Hartmann–Hahn (HH) matching profile into a centerband and additional bands of higher orders. The matching profiles differ with the substance categories. Therefore, signal intensity is usually lost, when e.g. the routine standard NH₄H₂PO₄ is used for optimizing the ¹H–³¹P HH match prior to measuring phosphines and their metal complexes in polycrystalline or immobilized form. Here, a variety of model compounds, such as Ph₂PCH₂CH₂PPh₂ and (CO)₂Ni(PPh₃)₂, which can be used as ³¹P CP standards for analogous substances or materials are presented. Investigating the influences of MAS frequency, contact time, ¹H pulse power and sample volume on the matching profiles of the model compounds leads to general trends. Thereby, a new strategy for measuring difficult samples with CP at high MAS rates has been developed: their optimum CP parameters are derived from the most intense maxima in the HH matching profiles of the corresponding model compounds. This new strategy is compared with variations of a conventional ramp sequence. Although the latter generally provide smaller signal half‐widths, the new strategy leads to higher signal intensities. The new method was successfully applied to polycrystalline and immobilized phosphines and catalysts. Copyright © 2003 John Wiley & Sons, Ltd.     

Diffusion and reaction in an immobilized enzyme starch saccharification catalyst

Effectiveness factors were predicted from measurements of basic parameters made on single oligosaccharides, and the prediction was compared to experimental effec tiveness factors for the reaction of each oligosaccharide in the immobilized enzyme catalyst. Kinetic parameters were obtained for the hydrolysis of each oligosaccharide catalyzed by soluble glucoamylase, and were fit with a subsite model equation capable of generalization to all sizes of oligosaccharide. Diffusion coefficients in free solution were determined from movement out of a capillary tube. Spatial characteristics of the immobilized enzyme bed were obtained from pulse response experiments, allowing the calculation of effective diffusivities. Experimental effectiveness factors plotted against modulus were in reasonable agreement with the predictions.     

Determination of intrinsic properties of immobilized enzymes

Staphylococcal nuclease has been insolubilized, directly through its amino groups, on CNBr-activated Sepharose 2B. For kinetic studies, a small substrate (thymidine 5′-(p-nitrophenyl phosphate) 3′-phosphate) has been used to measure the hydrolytic activity. With this system the absence of diffusional limitation has been proven. Eadie-Hofstee analysis of the data has been employed to determine the intrinsic kinetic constants of the insolubilized enzyme. Thek _cat-pH andK _M−pH profiles and the activation energies are similar for the soluble and for the insolubilized nuclease. At the same time conditions are established in which a stirred batch reactor containing particles of insolubilized nuclease behaves as an open system.     

功能材料在蛋白质组研究中的应用进展

随着对蛋白质组鉴定深度、定量准确性及分析速度越来越高的要求,对蛋白质组学方法的研发提出了新的挑战。为了应对这些挑战,传统的蛋白质组学方法因其灵敏度低、准确性差以及耗时长等不足已经难以满足蛋白质组学研究领域不断提出的新需求。而将通过光、电、磁、热、化学、生化等作用合成具有特定功能的材料用于蛋白质组的研究,可以克服传统蛋白质组学分析技术的局限性,为蛋白质组学研究起到促进作用。该文对功能材料在蛋白质组研究中应用的新进展进行综述。     

固定化胰蛋白酶整体小柱的制备及其用于微量蛋白质的快速酶解

发展了一种光引发聚合法制备固定化胰蛋白酶整体小柱的方法,以用于微量蛋白质的快速酶解。整体小柱由功能单体4-戊烯酸琥珀酰亚胺酯、甲基丙烯酸羟乙酯,交联剂季戊四醇三丙烯酸酯和三元致孔剂二甲基亚砜、N,N-二甲基甲酰胺、十二醇在20 μL的移液器吸头尖端原位聚合而成。形成整体柱后,胰蛋白酶分子通过氨基与琥珀酰亚胺酯反应实现固定化。系统研究了聚合溶液中活性酯含量与柱床体积对胰蛋白酶固载量的影响,评价了固定化酶整体小柱对标准蛋白细胞色素C和牛血清白蛋白的酶解效率,以及整体小柱的稳定性和重复性。结果表明,在离心辅助下,酶解过程可在10 min内完成,批次间具有良好的重复性。最后将固定化酶整体小柱应用于1×10⁵个人急性早幼粒白血病（NB4）细胞与人急性T细胞白血病（Jurkat T）细胞的快速酶解,经纳升级液相色谱与高分辨质谱联用分析后鉴定得到2489个和2572个蛋白质。相比于溶液状态下的酶解,分别提高了2.2%和6.1%的蛋白鉴定数量,展现了其在蛋白组学研究中的应用潜力。     

Screening and isolation of cyclooxygenase‐2 inhibitors from Trifolium pratense L. via ultrafiltration,enzyme‐immobilized magnetic beads,semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography

Nonsteroidal anti‐inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase‐2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase‐2 inhibitors can also act through cyclooxygenase‐independent mechanisms. In this study, using ultrafiltration, enzyme‐immobilized magnetic beads, high‐performance liquid chromatography, and electrospray‐ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase‐2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase‐2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography, and high‐speed counter‐current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.     

Lipophilicity and bio‐mimetic properties determination of phytoestrogens using ultra‐high‐performance liquid chromatography

This paper presents lipophilicity and bio‐mimetic property determination of 15 phytoestrogens, namely biochanin A, daidzein, formononetin, genistein, genistein‐4,7‐dimethylether, prunetin, 3,4,7‐trihydroxyisoflavon, 4,6,7‐trihydroxyisoflavon, 4,6,7‐trimethoxyisoflavon, daidzin, genistin, ononin, sissotrin, coumestrol and coumestrol dimethylether. High‐performance liquid chromatography with fast gradient elution and Caco‐2 cell line were used to determine the physicochemical properties of selected phytoestrogens. Lipophilicity was determined on octadecyl‐sylane stationary phase using pH 2.0 and pH 7.4 buffers. Immobilized artificial membrane chromatography was used for prediction of interaction with biological membranes. Protein binding was measured on human serum albumin and α‐1‐acid‐glycoprotein (AGP) stationary phases. Caco‐2 assay was used as a gold standard for assessing in vitro permeability. The obtained results differentiate phytoestrogens according to their structure where aglycones show significantly higher lipophilicity, immobilized artificial membrane partitioning, AGP binding and Caco‐2 permeability compared with glucosides. However, human serum albumin binding was very high for all investigated compounds. Furthermore, a good correlation between experimentally obtained chromatographic parameters and in silico prediction was obtained for lipophilicity and human serum albumin binding, while the somewhat greater difference was obtained for AGP binding and Caco‐2 permeability.     

Effective one-pot synthesis of tetrahydrobenzo[b]pyran derivatives using nickel Schiff-base complex immobilized on iron oxide nanoparticles

Nickel Schiff-base complex immobilized on silica-coated Fe₃O₄ as a heterogeneous catalyst was designed and characterized by different techniques, such as Fourier transform infrared (FT-IR), X-ray powder diffraction (XRD), field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), inductively coupled plasma (ICP) and vibrating sample magnetometry (VSM) thermogravimetric analysis (TGA), and Brunauer–Emmett–Teller (BET). The synthesized nanocatalyst has been explored as a new and efficient recyclable heterogeneous catalyst for the one-pot three-component synthesis of tetrahydrobenzo[b]pyran derivatives. The reaction proceeds smoothly to supply the respective products in excellent yields and low reaction times. The catalyst can be easily recovered by a magnetic field and reused for eight consecutive reaction cycles without significant loss of activity.