固定Fe 3＋的磁性微球分离富集鼠肝中铁结合蛋白的初步研究

采用键合Fe3＋的纳米材料分离富集了大鼠肝脏中的铁结合蛋白质组, 并进行了质谱分析. 在相同的起始富集蛋白质量以及相同的吸附和洗脱条件下, 键合了Fe3＋的磁性纳米材料比未键合金属离子的空白材料富集了更多的蛋白质, 经质谱鉴定得到42个蛋白质, 主要包括代谢酶类、呼吸链主要成员、氧化还原蛋白、转运蛋白、血红蛋白等.     

磁性纳米材料的制备及其吸附重铬酸根离子的性能

采用沉降法合成的Fe3O4,通过二氧化硅的包裹和3-氯丙基三甲基硅烷处理后键合聚乙烯亚胺(PEI)、季铵盐化等步骤合成功能化的磁性纳米材料。 二氧化硅的包裹增加了Fe3O4纳米颗粒的分散性和稳定性。 使用FT-IR、SEM、TG和振动样品磁强计对其进行了表征,结果表明,成功的改性了磁性纳米材料。 采用静态法研究了磁性纳米材料对重铬酸根的吸附性能及各种因素对吸附性能的影响。 分离富集环境中重铬酸根的适宜条件为：酸性介质,温度25 ℃,重铬酸钾的浓度0.8 g/L,吸附量120 mg/g,接枝量越大,吸附量增加。     

金属卟啉及其与咪唑络合物的电喷雾质谱研究

利用电喷雾电离质谱（ESI－MS）研究了3种金属卟啉化合物（MTPP＝MnTPP,TeTPP和CoTPP）,探讨了在这些化合物中苯取代基与卟啉环间的键合能力以及金属卟啉与咪唑的配位情况。研究结果表明,金属卟啉的外围取代基苯基与卟啉环的键合能力按Mn、Fe和Co的次序变弱。金属卟啉与咪唑形成的络合物的离子丰度随配体浓度的增加而增强;在相同的配体浓度下,络合物的离子丰度按Mn、Fe和Co顺序依次增加,其中,CoTPP络合物的稳定性最强。     

Fe3O4/TiO2 纳米材料靶上脱盐的基质辅助激光解析/电离质谱新方法研究

建立了一种简便、高效的靶上脱盐新方法。利用Fe3O4/TiO2磁性纳米材料对肽段的吸附作用,将其作为载体用于靶上肽段富集和脱盐。在对纳米材料使用量、浸洗条件进行优化的基础上,成功地鉴定了溶于10mol/L尿素溶液的100fmol的肌红蛋白样品,也对溶于3mol/L尿素溶液中的10fmol的肌红蛋白样品进行了成功地分析鉴定。通过对肌红蛋白样品进行预处理和质谱分析重复实验,表明该方法重现性好,且简便、高效,所需时间短,一次可同时处理多个样品,易于实现通量化,为有效地解决目前蛋白质组分析中所面临的基质辅助激光解析/离子化飞行时间质谱耐盐性差的问题提供了一种新的手段。     

功能化磁性纳米材料在磷酸化肽富集中的应用

蛋白质磷酸化是最重要和最普遍的翻译后修饰之一。基于质谱的技术已成为分析蛋白质磷酸化的重要手段。然而,磷酸化肽固有的低丰度和电离效率以及由非磷酸化肽共存引起的严重抑制使得直接质谱分析仍然是一个挑战。为解决此问题,需在质谱分析前对磷酸化蛋白质进行选择性富集。磁性纳米材料具有良好的磁响应性,可以在外界磁铁的帮助下实现与溶液的迅速分离。功能化磁性纳米材料作为一种新型的分析技术已在蛋白质组学研究中得到广泛的应用。该文就近年来对磁性纳米粒子进行各种功能化修饰以提高其特异性吸附能力的吸附材料在磷酸化肽的富集方面的应用予以综述,并展望了功能化磁性纳米材料在磷酸化肽富集领域的应用前景。     

A型流感病毒血凝素蛋白S-棕榈酰化修饰的质谱分析

蛋白质S-棕榈酰化修饰是指棕榈酸分子通过硫酯键共价结合在蛋白质分子的半胱氨酸( S)的巯基侧链上,是蛋白质脂类修饰的重要形式之一,在细胞信号转导、代谢等过程中起着重要作用。本实验首先利用酰基-生物素置换反应,将A型流感病毒血凝素蛋白上的S-棕榈酸分子转换为含有生物素( Buotun)分子的标签。生物素标记蛋白经特异性富集、电泳分离纯化后,进行胶内水解。再利用质谱技术对水解混合物进行分析。结果表明,经酰基-生物素置换方法处理A型流感病毒裂解产物后,蛋白质耦联生物素的浓度(羟胺处理,＋Hydroxylamune)与空白对照组(未加羟胺处理,-Hydroxylamune)的比值大于3;对经富集后的流感病毒血凝素蛋白进行了质谱分析,鉴定了 A 型的两个 S-棕榈酰化修饰位点,分别位于蛋白羧基末端的 Cys562和Cys565。本研究为大规模研究S-棕榈酰化修饰蛋白提供了一种特异、有效的分析方法。     

磷酸化蛋白质组学中的分离富集方法研究进展

蛋白质的磷酸化是一种可逆性的翻译后修饰,在细胞的增值、分化、信号转导以及转录与翻译调控、蛋白质复合体的形成、蛋白质降解等方面发挥着极为重要的作用.因此磷酸化蛋白的鉴定成为翻译后修饰研究的重要内容.但由于磷酸化蛋白的丰度较低, 难以用质谱直接检测.为了解决这个问题,改善质谱对磷酸肽的信号响应, 需要对磷酸化蛋白质或磷酸肽进行富集.本文系统地介绍了磷酸化蛋白组学研究中应用较为广泛和最新建立的各种分离富集方法的原理、特点、应用研究进展,包括抗体富集法、激酶特异富集法、亲和富集法、化学修饰法、多种色谱分离富集方法以及MALDI靶盘富集法.     

氧化锆微球担体-8-羟基喹啉鳌合固定相的合成及其在痕量金属分析中的应用

以氧化锆微球为担体,通过mannich反应得到在氧化锆微球上键合8-羟基喹啉的痕量金属富集分离固定相,并对该固定相的键合金属离子能力进行了研究。以合成的氧化锆微球键合8-羟基喹啉金属鳌合固定相用作微柱流动注射(FIA)在线分离、富集和电感耦合等离子体质谱(ICP-MS)分析流程中的固定相,对大洋海水中的痕量铅和镉进行了在线分析,得到了较满意的结果。     

基于天冬氨酸的固定化金属离子亲和色谱材料用于磷酸化肽选择性富集

采用点击化学的方法将自然界中的天冬氨酸(aspartic acid)键合到硅球上(命名为Click Asp),并将Fe3+配位到Click Asp上,合成固定金属离子亲和色谱(IMAC)材料(Fe3+-Click Asp);采用红外光谱、X射线电子能谱和扫描电镜等表征证明Fe3+-Click Asp成功合成。将此IMAC材料用于蛋白质酶解液和牛奶中的磷酸化肽的富集,实现了磷酸化肽的高选择性富集。本研究为磷酸化蛋白质组学提供了新材料和新方法。     

纳米颗粒预富集方法辅助质谱分析在蛋白质组学中的最新进展

由于生物样本中蛋白质含量的动态范围跨越多个数量级,快速、高效的蛋白质分离富集是低丰度蛋白质成功鉴定的关键.一些具有重要生物学意义的蛋白质的表达量通常很低,预富集就成了这些蛋白质获得成功质谱鉴定和分析的必备条件.富集的主要目的就是有选择性地从复杂生物样本中分离目标分子,从而达到降低样本复杂程度,辅助后续质谱高灵敏鉴定.近些年来,将纳米颗粒引入到这一领域大大促进了富集技术的发展.在本文中,我们集中介绍了近年来发展起来的,利用不同的纳米颗粒,富集低丰度和特定翻译后修饰的蛋白质或多肽并适合质谱鉴定的蛋白质预处理技术.     

Fe³⁺-immobilized nanoparticle-modified capillary for capillary electrophoretic separation of phosphoproteins and non-phosphoproteins

A fused-silica capillary modified with Fe3?-immobilized magnetic nanoparticles (Fe3?-IMAN) has been investigated for the capillary electrophoretic (CE) separation of phosphoproteins and non-phosphoproteins. The Fe3?-IMAN capillary was achieved by covalently immobilising epoxy-based magnetic silica nanoparticles (160?nm) on the prederivatized 3-aminopropyl-trimethoxysilane (APTMS) fused-silica capillary (75?μm id), followed by disodium iminodiacetate and Fe3?. The buildup process was examined by measuring the streaming potentials of the bare capillary, APTMS capillary, epoxy-based nanoparticle capillary and Fe3?-IMAN capillary by varying the buffer pH. An inverted fluorescence microscope was used to determine the surface features of the Fe3?-IMAN capillary derivatized with morin. Further experimental results confirmed that Fe3?-IMAN bonded on the inner wall of the APTMS capillary could provide sufficient solute-bonded phase interactions to allow for the CE separation of phosphoproteins and non-phosphoproteins at concentration levels down to 50?μg/mL. The highest number of theoretical plates obtained was about 233,000/m, and the relative standard deviation (RSD) for migration times was <2.57% for eight consecutive runs, respectively. Additionally, the Fe3?-IMAN modifing method was also applied to the analyses of bovine milk proteins. With simplicity, high resolving power, and high repeatability, the proposed method has shown great potential for phosphoproteomics applications.     

Copper removal by carbon nanomaterials bearing cyclam-functionalized silica

The synthesis of metal (Fe, Co, Ni)-encapsulated carbon nanomaterials coated with cyclam-bonded silica has been described. The organic layer was identified by Fourier transform infrared spectroscopy and elemental analysis. The functionalized magnetic nanomaterials were employed to extract the divalent cations: copper, calcium, cobalt, manganese and nickel from aqueous solutions. Their adsorption capacities were studied by the batch procedure. The concentration of cations extracted was determined by inductively coupled plasma mass spectrometry. Influence of different parameters viz. pH, amount of the compound studied, contact time, on the cation extraction was investigated. Under optimum conditions copper extraction was significantly more efficient when compared with other coexisting ions.      

Fe3O4@Al2O3 magnetic core-shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres with phosphopeptides has been developed. With a well-defined core-shell structure, the Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres over Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC (immobilized metal affinity chromatography) resin, and TiO(2) beads. Finally, the Al(2)O(3) coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.     

Fungus-mediated biotransformation of amorphous silica in rice husk to nanocrystalline silica

Rice husk is a cheap agro-based waste material, which harbors a substantial amount of silica in the form of amorphous hydrated silica grains. However, there have been no attempts at harnessing the enormous amount of amorphous silica present in rice husk and its room-temperature biotransformation into crystalline silica nanoparticles. In this study, we address this issue and describe how naturally deposited amorphous biosilica in rice husk can be bioleached and simultaneously biotransformed into high value crystalline silica nanoparticles. We show here that the fungus Fusarium oxysporum rapidly biotransforms the naturally occurring amorphous plant biosilica into crystalline silica and leach out silica extracellularly at room temperature in the form of 2-6 nm quasi-spherical, highly crystalline silica nanoparticles capped by stabilizing proteins; that the nanoparticles are released into solution is an advantage of this process with significant application and commercial potential. Calcination of the silica nanoparticles leads to loss of occluded protein and to an apparently porous structure often of cubic morphology. The room-temperature synthesis of oxide nanomaterials using microorganisms starting from potential cheap agro-industrial waste materials is an exciting possibility and could lead to an energy-conserving and economically viable green approach toward the large-scale synthesis of oxide nanomaterials.     

Chiral Diamine/Brønsted Acid Conjugates Confined in Mesoporous Silica as Catalyst for the Asymmetric Aldol Reaction

Chiral primary-tertiary diamine/Brønsted acid conjugates were selectively immobilized on the inner surface of mesoporous silica and used as catalyst for the asymmetric aldol reaction of acetone with various aldehydes. The catalyst showed modest reactivity and enantioselectivity and can be reused 6 times without loss of activity and enantioselectivity. As compared with silica gel as the support, an increase in ee value of the reaction product was observed with the SBA-15-immobilized organic catalyst. This was believed to be due to the confinement effect of the nanopores.     

Preparation of hybrid mesoporous silica luminescent nanoparticles with lanthanide(III) complexes and their exhibition of white emission

We chose dipicolinic acid as a tridentate chelating unit featuring ONO donors to react with lanthanide(III) ions to yield tight and protective N(3)O(6) environments around the lanthanide(III) ions. We immobilized the lanthanide(III)-dipicolinic acid complexes on colloidal mesoporous silica with diameter smaller than 100 nm by a covalent bond grafting technique and obtained nearly monodisperse luminescent Eu-dpa-Si and Tb-dpa-Si functionalized hybrid mesoporous silica nanomaterials. These hybrid nanomaterials were characterized by powder X-ray diffraction, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, nitrogen adsorption-desorption, and photoluminescence spectroscopic techniques. The hybrid mesoporous silica nanoparticles exhibit intense emission lines upon UV-light irradiation, owing to the effective intramolecular energy transfer from the chromophore to the central lanthanide Eu(3+) and Tb(3+) ions. Furthermore, the functionalized nanomaterials can be turned to white light materials after annealing at high temperature.     

Fluorescence immunosensor based on p-acid-encapsulated silica nanoparticles for tumor marker detection

A novel, simple and highly sensitive amplified fluorescence label-free immunosensor by using p-acid-encapsulated silica nanomaterials has been developed for the first time. 4,4'-(2,5-Dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl)dibenzoic acid (p-acid) and p-acid-encapsulated silica were prepared, and characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, ultraviolet visible spectroscopy (UV-vis) and fluorescence spectroscopy. In layer-by-layer self-assembling processes using (3-aminopropyl)triethoxysilane, p-acid@SiO(2) was assembled on the glass substrate. Antibody was immobilized on the surface of p-acid@SiO(2) with N,N'-carbonyldiimidazole. The functional nanomaterials present good analytical properties with a calibration range of 0.1-100 ng mL(-1), and allow the detection of carcinoembryonic antigen (CEA) at a concentration as low as 0.04 ng mL(-1). What is important is that the as-synthesized p-acid@SiO(2) nanomaterials could be further extended for the detection of other biomarkers or biocompounds.     

Cu-Fe基双孔载体催化剂结构和低碳醇合成反应性能

采用超声浸渍法制备了Cu、Fe 双活性组元改性的双孔载体(M)催化剂, 采用N₂物理吸附、H₂程序升温 还原/脱附(H₂-TPR/TPD)、X射线衍射(XRD)等表征手段考察了催化剂中Cu-Fe的相互作用, 并在固定床反应器 中评价了Cu/Fe摩尔比的改变对低碳醇合成反应性能的影响. 结果表明: 小孔硅溶胶浸渍在大孔硅凝胶中可形 成具有不同纳米孔径结构的双孔载体, 增加小孔硅溶胶的含量可促使双孔载体中小孔纳米结构尺寸变小. Fe/ Cu摩尔比的增加有利于铜物种在载体表面的分散, 促进了表层CuO和Fe₂O₃的还原, 加强了双孔载体内孔道 与铜铁氧化物之间的相互作用, 促使了单质铜的分散和铁碳化物的生成. CO加氢反应活性和低碳醇时空收率 随着Fe/Cu 摩尔比的逐渐增加呈现增加的变化趋势. 当Fe/Cu 摩尔比增加到30/20 时, Cu-Fe 基双孔载体催化 剂的CO转化率增加到46%, 低碳醇的时空收率增加到0.21 g·mL^-1·h^-1, C₂₊OH/CH₃OH质量比达到1.96.