亲和标记-基质辅助激光解吸电离飞行时间质谱用于蛋白质相对定量方法的研究

将亲和标记技术和基质辅助激光解吸电离飞行时间串联质谱联用,建立了蛋白质相对定量方法,以牛血清白蛋白为实验对象,考察了该方法的准确度、重现性等指标;又以数种标准蛋白混合物为研究对象,考察了该方法的动态范围及相应的标准偏差,为实际生物样品中差异蛋白质分析奠定了基础。     

Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate-polyacrylamide gels

Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.     

Self-assembled luminescent CdSe-ZnS quantum dot bioconjugates prepared using engineered poly-histidine terminated proteins

We report a simple and versatile approach for the conjugation of luminescent CdSe-ZnS core-shell quantum dots (QDs) to proteins through coordination of engineered C-terminal oligohistidine sequences. Several histidine tail containing proteins were self-assembled onto the QD surface using this method. A recombinant antibody specific for the high explosive 2,4,6-trinitrotoluene (TNT) was conjugated to QDs through a carboxy terminal histidine tail and the bioconjugate used to detect TNT by competitive immunoassay. TNT was detected over the range of 10 μg/ml down to 41 ng/ml using the scFv conjugated to QDs. These results open up the possibility to conjugate luminescent QDs to a whole range of proteins to form QD bioconjugates that can be effectively used in bio-oriented applications, such as sensing, imaging, immunoassay and other diagnostics.     

液相组合合成的纯化方法

综述了近8年来液相平行合成和组合合成中应用的不同技术, 包括可溶性载体、氟合成技术、离子液体、固相试剂树脂以及低聚乙烯二醇(OEG)衍生物的应用等几方面内容. 论述了它们的基本原理以及相关的应用实例, 并着重强调了目标化合物的分离纯化方法.     

Sensitive Voltammetric Sensing of the 2,3‐Dimethyl‐2,3‐dinitrobutane (Dmnb) Explosive Taggant

The highly sensitive voltammetric detection of the 2,3‐dimethyl‐2,3‐dinitrobutane (DMNB), a required additive to commercial plastic explosives, is described. The protocol relies on a fast square‐wave voltammetric measurement of the DMNB explosive taggant at an unmodified carbon fiber electrode using a phosphate buffer (pH 7.0) solution. Different solutions and working electrodes were evaluated. Under the optimal conditions, a linear response is observed over the 300–3000 μg/L DMNB concentration range examined, with a detection limit of 60 μg/L. A highly stable response, with a relative standard deviation (RSD) of 2.6%, is observed for 30 repetitive measurements. Such electrochemical approach offers great promise for a simple, rapid, sensitive and inexpensive field screening of plastic explosives. Preliminary data illustrate the utility of electrochemical detection for electrophoretic microchips for the simultaneous measurements of DMNB, cyclotrimethylenetrinitramine (RDX) and pentaerythritoltetranitrate (PETN).     

Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry

We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.     

Preparation of core–shell structure Fe3O4@SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His‐tag protein purification

The core–shell structure Fe₃O₄/SiO₂ magnetic microspheres were prepared by a sol–gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu²⁺, Ni²⁺ and Zn²⁺, were chelated on the Fe₃O₄@SiO₂–IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni²⁺‐chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe₃O₄@SiO₂–IDA–Ni²⁺ magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His‐tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd.     

Concise Synthesis of Photocleavable Molecular Tag for Laser Desorption Ionization Mass Spectrometry via Fries Reaction

Abstract

A new synthetic route for the photocleavable molecular tag for laser desorption ionization mass spectrometry (LDI-MS) was achieved using the Fries reaction of 2,6-dimethylphenyl ester as its key reaction. Zirconium chloride was used as uniquely efficient adjuvant to promote the reaction. The molecular tag was obtained in five steps without chromatographic purification.

Supplemental materials are available for this article. Go to the publisher's online edition of Synthetic Communications® to view the free supplemental file.     

供应链集中决策下RFID技术应用决策分析

RFID技术可以对供应链系统中的产品信息进行有效追溯和实时共享,以化解系统管理效率、安全防范、监控和保障能力极其低下等问题.但是,由于应用成本的制约,使很多附加值较低的行业仍望而却步,严重制约了RFID技术的发展和普及.以单个零售商和供应商组成的供应链系统为研究对象,首先,对基于RFID的供应链产品入库及日常盘点管理流程进行了简要分析;在此基础上,分别构建了集中决策下采用与不采用RFID技术时的供应链利润模型,并对其最优决策问题进行详细分析,得出供应链系统在集中决策下实施RFID技术的投资决策条件,所得结论可以为供应链系统在集中决策下实施RFID技术应用决策问题提供理论支持.