液相色谱-质谱联用分析流感病毒感染引起宿主蛋白类泛素化修饰

干扰素刺激基因15编码蛋白质(Interferon stimulated gene 15 kDa protein, ISG15)是最早被鉴定的类泛素分子蛋白质,在病毒感染和免疫调节等方面具有重要作用。本研究利用免疫沉淀技术将被类泛素 ISG15修饰的蛋白富集纯化,采用液相色谱-质谱联用技术对流感病毒感染 A549宿主细胞过程中产生的类泛素 ISG15修饰蛋白进行了分析。实验结果表明,在流感病毒感染的实验组 A549细胞中,鉴定到了22种来源于宿主细胞的ISG15修饰的蛋白,包括类泛素蛋白 ISG15、细胞周期蛋白-T1、热休克蛋白71、钙调素结合蛋白、真核翻译起始因子等,以及1种来源于流感病毒的非结构蛋白 NS1。在鉴定的22种宿主蛋白中,有6种蛋白在未感染病毒的对照组 A549细胞中也得到鉴定,包括膜联蛋白 A1、果糖二磷酸醛缩酶 A、线粒体三磷酸腺苷合成酶亚基 g、烯醇化酶、肌动蛋白、微管蛋白。生物信息学分析表明,流感病毒感染引起的 ISG15修饰的宿主蛋白分别归属于9个不同的蛋白分类,包括细胞骨架蛋白、分子伴侣蛋白、酶调节剂、核酸结合蛋白、激酶类、转移酶类、转录因子、氧化还原酶类以及结构蛋白。本研究为大规模分析鉴定 ISG15修饰蛋白提供了一种特异、有效的研究方法。     

稀土和微量元素对鸡胚生长发育影响的研究

研究了稀土和微量元素对鸡胚生长发育的影响及吸收分布情况。以海兰种蛋为实验材料,在稀土中按一定比例添加不同量的7种生物必需微量元素,注入种蛋进行孵化,实验发现,稀土与微量元素共用对鸡胚生长发育有一定影响;稀土剂量一定,加入不同量的微量元素,稀土和微量元素会产生最佳配比;最佳配比能使鸡胚正常生长发育,且稀土在各组织中的积累较使用同剂量的单一稀土少。     

MAT—251质谱计^1^5N相对测量方法的误差分析

本文讨论了用ＭＡＴ－２５１质谱计进行１５Ｎ天然丰度测量的过程中，各个环节和整个方法的精度，并与国外同类工作的测量结果进行了比较。结果表明：本方法的精度和准确度均达到国际上的先进水平。     

原油中含氧化合物的质谱分析

本文介绍自原油分离含氧化合物,酯化混合酸和用毛细管气相色谱-质谱(GC-MS)鉴定其组分的方法。色谱-质谱分离并鉴定出C_8—C_(30)的脂肪酸、分子量为484和498的二对藿烷酸的立体异构体,烷基酚,邻苯二甲酸酯等,并首次发现石油中含有癸二酸二异辛酯类组分。     

可溶性耐药相关钙结合蛋白的生物质谱分析与鉴定

可溶性耐药相关钙结合蛋白最初由Meyers等从长春新碱诱导的中国仓鼠多药耐药细胞株中发现,在多种耐药细胞株中均有高表达,它作为一种钙结合蛋白,一旦大量表达,即可改变细胞内钙环境,从而影响信号传导,使其它受钙调节的蛋白质的功能发生改变。     

烟草中5种糖苷类化合物的液相色谱-质谱联用分析

采用反相高效液相色谱-质谱(HPLC-MS)联用技术分析了烟草提取液中的5种糖苷类化合物.将烟叶烘干、磨碎后用95%乙醇提取,提取液用正己烷-乙醚(体积比1∶1)除去油脂,水层经大孔树脂分离除杂后,进HPLC-MS分析,获得了5种糖苷类化合物的结构信息.通过对相对分子质量及碎片离子结构信息的解析,推测5种糖苷类化合物分别为:α-紫罗兰醇-β-D-葡萄糖苷、3-氧代-α-紫罗兰醇-β-D-葡萄糖苷、4-羟基-α-紫罗兰醇-β-D-葡萄糖苷、3-羟基-β-大马酮-β-D-葡萄糖苷和地黄普内酯-β-D-葡萄糖苷.     

高效液相色谱／质谱分析磨菇柄中5‘—核苷酸

采用高效液相色谱/质谱法，对蘑菇柄中5‘-核苷酸进行了分析。在C18柱上，以含0.05%磷酸的水-甲醇（95/5，V/V）为流动相进行了分离，检测波长260nm。以HPLC与LC/MS复合定性磨菇柄中5‘-核苷酸为5‘-鸟苷酸、5‘-腺苷酸和5‘-胞苷酸。采用外标法定量，它们的回收率分别为98.9%、95.1%、94 .1%和101.4%，检测限分别为2.8、2.7、3.5和4.3mg/L。     

质谱的无机痕量分析进展

概述了电感耦合等离子体质谱(ICP-MS)、激光电离质谱(LIMS)和共振电离质谱(RIMS)、火花源质谱(SSMS)、辉光放电质谱(GDMS)、二次离子质谱(SIMS)、热电离同位素稀释质谱法(ID-TIMS)等无机质谱分析方法的基本原理、方法特点和应用进展,概括了ICE-MS和IDMS方法的优缺点;采用溶液进样的ICP-MS和TIMS稀释法,给出的测量结果不确定度要高于固体进样的质谱法。     

辉光放电质谱分析中质谱干扰及其校正方法的现状

详细地介绍了辉光放电质谱分析中的质谱干扰如同量异位素、多原子离子和多电荷离子干扰。从同位素选择、高分辨率仪器、碰撞诱导解离、离子源冷却、数学方法校正、放电气体更换和放电气体纯度提高等方面对辉光放电质谱的质谱干扰校正方法的现状进行了评述(引用文献共68篇)。     

TiO2对流感病毒(H1N1)灭活作用的研究

利用滴度测定和透射电镜观察研究了365 nm的紫外光照射下TiO2对流感病毒(H1N1)的灭活性能, 并结合催化剂样品的XRD分析、 N2气吸附性能测定及其在实验条件下的表面Zeta电势的测量结果, 探讨了催化剂用量、 焙烧温度、 比表面积以及表面电性与灭活性能的关系. 研究结果表明, 400 ℃时焙烧的TiO2对H1N1的灭活性最好; TiO2的表面电性对灭活性有显著影响; TiO2对H1N1的光催化灭活作用首先发生在H1N1的纤突部分, 纤突部分的破坏导致H1N1的失活, 分解直至矿化.      

乙酰化蛋白的质谱鉴定研究

报道了两种生物质谱技术ESI-MS和MALDI-MS在鉴定乙酰化修饰蛋白BSA-ac中的应用研究结果. 乙酰化修饰蛋白通过特征碎裂峰m/z 126.1或MS/MS质谱图中相差一个赖氨酸的相邻b或y离子之间170 Da分子量的差异确证赖氨酸乙酰化修饰, 并且后者提供具体修饰位点信息. 研究提示ESI-MS和MALDI-MS两种质谱技术均可用于鉴定实际复杂样品中的乙酰化蛋白, 且在乙酰化蛋白的鉴定中各有其优点.     

单管高灵敏度等温扩增技术快速检测甲型H1N1流感病毒

建立了单管逆转录环介导等温扩增法(RT-LAMP)快速检测甲型H1N1流感病毒的方法.针对甲型H1N1流感病毒的M基因和HA基因的保守区,设计了两组特异性引物,分别用于筛选甲型流感病毒及鉴定甲型H1N1流感病毒.对反应体系中的关键因素进行优化,反应结果可直接通过浊度或者SYBR Green Ⅰ荧光进行判定.本方法最低可...     

Screening of Proteins Interacting with Nonstructural 1 Protein of H5N1 Avian Influenza Virus from T7-phage Display Library

Avian influenza virus(AIV) nonstructural 1(NS1) gene was amplified by real-time polymerse chain reaction( RT-PCR) and inserted into pET28a, then transformed into E. coli BL21(DE3) competent cell. With the induction of isopropyl-β-D-thiogalactoside(IPTG) and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA library. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene- Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1(NOLC1) and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other methods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for further studying biological function of NS1 protein.     

检测禽流感H5亚型病毒的阻抗型免疫研究

研制了一种可用于H5亚型禽流感病毒快速检测的阻抗型免疫传感器。通过蛋白A将H5N1表面抗原血凝素(HA)的单克隆抗体固定于金叉指阵列微电极表面,并与待测溶液中的目标抗原H5N1进行免疫反应。在[Fe(CN)6]3"/4"溶液中进行电化学阻抗谱扫描,表征电极的表面修饰及抗原捕获过程。当H5N1病毒浓度在21～26 HA unit/50μL范围时,其浓度的对数值与叉指阵列微电极的电子传递阻抗的变化值呈线性关系,相关系数为0.9885;检出限为20 HA unit/50μL,检测时间为1 h。此传感器特异性好,灵敏度高,可以重复使用,在病原微生物快速检测领域具有良好的应用前景。     

Identification of myosin heavy chain isoforms in porcine longissimus dorsi muscle by electrophoresis and mass spectrometry

Myosin heavy chain (MHC) isoforms have been considered as makers for muscle fiber types in relation to meat quality, whereas MHC isoforms in porcine skeletal muscle have not been fully identified. The improved technique of SDS‐PAGE and 2DE were used to separate porcine MHC isoforms. Western blotting with monoclonal antibodies including BA‐F8 (anti‐MHC slow/I), SC‐71 (anti‐MHC 2a and 2x), 10F5 (anti‐MHC 2b), and BF‐35 (anti‐MHC slow/I and 2a) and MS were used to confirm MHC migration rate and identify MHC isoforms from separated bands and spots. Up to 45% w/v of glycerol, 8% w/v of acrylamide content, and 25 h of electrophoretic time at 70 V allowed a clear separation of MHC isoforms. Major MHC isoforms such as slow, 2a, 2x, and 2b were clearly separated by SDS‐PAGE. A total of 23 MHC spots were separated and identified by 2DE and MS. Therefore, four MHC isoforms such as slow/I, 2a, 2x, and 2b could be identified by the improved SDS‐PAGEtechnique, 2DE and MS. Therefore, these techniques allow more accurate and accessible analysis in muscle fiber typing and in relationship between MHC isoforms, muscle fiber characteristics, and pork quality.     

Electrochemical Magnetoimmunosensing Approach for the Sensitive Detection of H9N2 Avian Influenza Virus Particles

A novel electrochemical magnetoimmunosensor for fast and ultrasensitive detection of H9N2 avian influenza virus particles (H9N2 AIV) was designed based on the combination of high‐efficiency immunomagnetic separation, enzyme catalytic amplification, and the biotin–streptavidin system. The reusable, homemade magneto Au electrode (M‐AuE) was designed and used for the direct sensing. Immunocomplex‐coated magnetic beads (IMBs) were easily accumulated on the surface of the M‐AuE to obtain the catalytically reduced electrochemical signal of H₂O₂ after the immunoreaction. The transducer was regenerated through a simple washing procedure, which made it possible to detect all the samples on a single electrode with higher reproducibility. The magnetic‐bead‐based electrochemical immunosensor showed better analytical performance than the planar‐electrode‐based immunosensor with the same sandwich construction. Amounts as low as 10 pg mL^?1 H9N2 AIV could be detected even in samples of chicken dung. This electrochemical magnetoimmunosensor not only provides a simple platform for the detection of the virus with high sensitivity, selectivity, and reproducibility but also shows great potential in the early diagnosis of diseases.     

Enrichment and Identification of Metallothionein by Functionalized Nano-Magnetic Particles and Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

As a low molecular weight protein with the ability of binding metal ions and high inducibility, metallothionein (MT) is often regarded as an important biomarker for assessment of heavy metal pollution in water environment. In the light of that the traditional process of enrichment and identification is time-consuming and complicated, we prepared a core-shell nanoparticle, gold-coated iron oxide nanoparticles (Fe₃O₄@Au NPs) herein. It possessed the advantages of fast response to magnetic fields and optical properties attributing to Fe₃O₄ and Au nanoparticles, respectively. The Fe₃O₄@Au nanoparticles could be used to enrich MT simply through Au–S interaction, and the purified proteins were determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The results showed that the Fe₃O₄@Au nanoparticles could directly enrich MT from complex solutions and the detection limit could be as low as 10 fg mL^?1.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。