水热法制备质谱专用提取多肽的金属螯合纳米磁珠

应用水热法合成质谱专用提取血清多肽的金属螯合纳米磁珠, 可用于质谱对血清中多肽分布情况的研究, 获得血清多肽谱. 对磁珠进行透射电镜(TEM), 原子力显微镜(AFM)和傅立叶红外光谱FT-IR的表征, 显示该粒子粒径在70～90 nm. 并通过质谱验证该金属螯合磁珠能有效提取血清中的多肽, 该磁珠为质谱进行疾病诊断解决样品制备的技术难题, 具有广阔应用前景.     

自动化磁珠提取纯化仪器快速检测禽流感

不久前,赛默飞世尔科技推出产品King Fisher系列自动化磁珠提取纯化仪器。据悉,此产品是联合Invitrogen公司Ambion病毒RNA提取试剂盒,将传统方法的禽流感检测由21天缩短至4h左右。此技术经过美国国家兽医服务实验室验证,是美国农业部认可的禽流感检测的分子生物学方法。禽流感快速高通量检测新技术是基于分子生物学的方法,在病毒感染的初期即可快速确诊病毒的存在。该方法包括两大主要步骤:(1)联合使用赛默飞世尔公司的King Fish-er自动化磁珠提取纯化仪器,及Invitrogen公司的Ambion病毒RNA提取试剂盒,进行自动化的病毒RNA提取,该步骤需时约30min;(2)实时定量RT-PCR检测病毒RNA,该步骤需时约3h。整体方法经过优化,并经过超过200000禽类咽喉拭子样品的检测验证。这项技术可以用于从生物体液和无细胞样品中(如血清、血浆、口腔拭子和细胞培养液)提取病毒RNA。从无细胞样品中分离纯化RNA比从组织和培养细胞中分离纯化RNA更具挑战性。自动化磁珠提取纯化仪器快速检测禽流感@林     

两种软件分析唾液多肽组结果的比较

以10个唾液样本为例,唾液经氧化石墨烯-磷酸镧纳米复合材料(LaGM)方法分离和高分辨串联飞行时间质谱进行多肽鉴定后,使用Peaks studio 8.5(PS)和Protein Pilot Software 4.0(PP)两种软件分别进行搜库分析后对比鉴定结果。研究发现,两种软件鉴定出的阈值以上肽段数量存在差异, PS软件通常能够鉴定出更多阈值以上肽段数目;两种软件鉴定出的阈值以上相同肽段在PS中占30%～60%,在PP中占60%～90%,即某些肽段只能被PS或PP一种软件所鉴定。但是,两种搜库结果在降解蛋白质数目上无明显规律,数目因样本而异。值得注意的是,如果以PP和PS的阳性结果相互参照,发现在一种软件中阈值以下的肽段,在另一种软件中也可能是在阈值以上,而且此概率与肽段的打分呈正相关。研究还发现,两种软件对不同长度肽段的鉴定有一定的偏好性:PS鉴定结果中短肽段较多,而PP软件可以给出更多的长肽段。比较而言,在PP中,短肽段易出现假阴性,在PS中,长肽段易出现假阴性,而信号的相对强度与阈值无明显的相关性。本研究结果表明,只使用一种软件进行分析,结果不能准确地代表多肽组的全部情况,两...     

唾液中大麻毒品液相小体积提取HPLC检测

建立了小体积液相提取HPLC测定唾液中大麻类毒品含量的方法。在pH=4缓冲溶液中,加入1mL含大麻标准品的家兔唾液试样,用0.5mL氯仿超声振荡提取5min,4000r/min离心10min,取下层液体挥干,用1mL乙腈溶解后进行HPLC分析。唾液中四氢大麻酚(THC)、大麻二酚(CBD)、大麻酚(CBN)、四氢大麻酚酸的检出限(3S/N)分别为16ng、10ng、11ng、10 ng,标准曲线线性范围分别为0.32μg/μL～3.20μg/μL、0.10μg/μL～1.00μg/μL、0.11μg～1.10μg/μL、0.20μg/μL～2.00μg/μL。平均回收率均在95%～105%之间。相对标准偏差(n=6)均在3%以内。     

表面电性可控磁珠微流控芯片在DNA提取中的应用

制备了表面电性可控的氨基化SiO_2@Fe_3O_4磁性复合微球,采用激光刻蚀和热压键合的方法制作了聚甲基丙烯酸甲酯(PMMA)材质的DNA固相萃取芯片,将磁珠灌注于芯片通道中,借助永磁铁固定并控制磁珠,将磁珠芯片应用于人类全血中的基因组DNA提取,优化了提取实验条件,并对提取产物进行凝胶电泳和PCR分析。实验结果表明,磁珠微流控芯片成功地从全血中提取出纯度较高的基因组DNA,提取效率约35%,提取液的凝胶电泳条带与商品化试剂盒提取的基因组DNA一致,提取液可用于进一步的PCR反应。     

用于磁珠法核酸提取的微流控芯片及自动化平台

基于磁珠法核酸提取原理,设计并制作出旋转驱动式核酸提取微流控芯片及自动化平台。微流控芯片包括裂解腔、清洗腔以及洗脱腔等结构,步进电机带动微流控芯片旋转,通过电磁铁吸附微流控芯片内的磁珠,实现磁珠在各腔室转移,完成核酸提取和纯化。对芯片表面疏水性、磁力大小、磁珠分散程度以及核酸洗脱时间进行优化。结果表明,当磁力大小为250 N时,可实现磁珠转移;磁铁放置于芯片上方1 mm时,腔室内磁珠分散程度最好。洗脱时间为20 min时,芯片上提取大肠杆菌的核酸浓度较高。微流控芯片与磁珠核酸提取技术相结合提取的核酸样本,可直接应用于后续聚合酶链式反应扩增环节,有利于实现核酸自动提取及扩增的一体化。     

姜黄素的提取方法研究

借助微波辅助手段,加入新制的系列咪唑类离子液体(ILs),利用正交法设计实验并优化了不同因素水平下生姜中姜黄素的提取工艺,利用其对1,1-二苯基-2-三硝基苯肼(DPPH)的清除率检测了姜黄素的抗氧化性.最佳提取工艺条件为:离子液体(溴化1-甲基-3-正癸基咪唑)浓度为1.5mol·L~(-1),乙醇体积分数为80%,料液比为1∶15,微波功率为165 W,微波时间为20 min,最终提取率为0.426%.此条件下姜黄素对DPPH自由基的清除率为65.07%,IC50为52.8μg·L~(-1),表明姜黄素有较强的抗氧化性.     

金银花绿原酸提取方法研究

采用高效液相色谱法测定金银花中绿原酸的含量,通过热回流法优化金银花绿原酸提取条件并进行正交试验分析. 试验结果表明,金银花中绿原酸最佳提取条件为乙醇体积分数80%、提取温度100 ℃、料液比1∶20 （g/mL）、提取时间2 h时,绿原酸提取率为7.1%. 方法分析准确、操作简便、设备要求低,可用作金银花的质量控制和开发利用.     

211At标记蛋白质或多肽的方法研究

α核素211At具有良好的辐射生物学性质,以对肿瘤细胞具有高亲和性的单克隆抗体或多肽类配体为载体则是实现211At肿瘤靶向治疗的最理想方式之一。本文介绍了211At标记蛋白质或多肽的方法的现状与进展,对存在的一些问题及今后的发展方向进行了讨论。     

质谱分析福尔马林固定组织的多肽谱

福尔马林固定组织时,甲醛与蛋白质多肽链的氨基酸侧链上的功能基团,特别是氨基、亚氨基、酰氨基、羟基和巯基等相结合,使蛋白分子间形成大分子网络,蛋白不再发生迁移达到固定目的[1].     

磁性珠状纤维素亲和吸附剂的制备与应用

采用反相悬浮包埋技术制备了粒径小于300um、粒径分布窄和湿态孔度高(85%～90%)的高顺磁性珠状纤维素,经高碘酸钠活化后,与具有生物活性的绒毛膜促性腺激素偶联,得磁性亲和吸附剂(每克磁性珠状纤维素上固载300～400IU绒毛膜促性腺激素).     

A Generic Multiple Reaction Monitoring Based Approach for Plant Flavonoids Profiling Using a Triple Quadrupole Linear Ion Trap Mass Spectrometry

Flavonoids are one of the largest classes of plant secondary metabolites serving a variety of functions in plants and associating with a number of health benefits for humans. Typically, they are co-identified with many other secondary metabolites using untargeted metabolomics. The limited data quality of untargeted workflow calls for a shift from the breadth-first to the depth-first screening strategy when a specific biosynthetic pathway is focused on. Here we introduce a generic multiple reaction monitoring (MRM)-based approach for flavonoids profiling in plants using a hybrid triple quadrupole linear ion trap (QTrap) mass spectrometer. The approach includes four steps: (1) preliminary profiling of major aglycones by multiple ion monitoring triggered enhanced product ion scan (MIM-EPI); (2) glycones profiling by precursor ion triggered EPI scan (PI-EPI) of major aglycones; (3) comprehensive aglycones profiling by combining MIM-EPI and neutral loss triggered EPI scan (NL-EPI) of major glycone; (4) in-depth flavonoids profiling by MRM-EPI with elaborated MRM transitions. Particularly, incorporation of the NH3 loss and sugar elimination proved to be very informative and confirmative for flavonoids screening. This approach was applied for profiling flavonoids in Astragali radix (Huangqi), a famous herb widely used for medicinal and nutritional purposes in China. In total, 421 flavonoids were tentatively characterized, among which less than 40 have been previously reported in this medicinal plant. This MRM-based approach provides versatility and sensitivity that required for flavonoids profiling in plants and serves as a useful tool for plant metabolomics.

Impurity Profiling of Dirithromycin by Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry

The aim of this study was to characterize as much as possible the unknown peaks in the chromatogram obtained with a non-volatile LC-UV system, which was published earlier for the separation of dirithromycin and its related substances. For this purpose, each peak eluting from the non-volatile system was collected and transferred to a MS, after performing a desalting process. The desalting procedure uses a XTerra RP C₁₈ column (250 mm x 4.6 mm, 5 µm) and two mobile phases consisting of a mixture of water / 0.1% (v/v) formic acid and a mixture of acetonitrile / 0.1% (v/v) formic acid, respectively. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ionization source (ESI), operating in the positive ion mode. In addition to the thirteen already known compounds, seven new compounds were elucidated. Five impurities showed modifications at the amino group of the desosamine molecule, one an alteration at position C-9 and one a modification at position C-13 of the macrolide ring.     

A Fast and Selective Approach for Profiling Vicinal Diols Using Liquid Chromatography-Post Column Derivatization-Double Precursor Ion Scanning Mass Spectrometry

Vicinal diols are important signaling metabolites of various inflammatory diseases, and some of them are potential biomarkers for some diseases. Utilizing the rapid reaction between diol and 6-bromo-3-pyridinylboronic acid (BPBA), a selective and sensitive approach was established to profile these vicinal diols using liquid chromatography-post column derivatization coupled with double precursor ion scan-mass spectrometry (LC-PCD-DPIS-MS). After derivatization, all BPBA-vicinal-diol esters gave a pair of characteristic isotope ions resulting from ⁷⁹Br and ⁸¹Br. The unique isotope pattern generated two characteristic fragment ions of m/z 200 and 202. Compared to a traditional offline derivatization technique, the new LC-PCD-DPIS-MS method retained the capacity of LC separation. In addition, it is more sensitive and selective than a full scan MS method. As an application, an in vitro study of the metabolism of epoxy fatty acids by human soluble epoxide hydrolase was tested. These vicinal-diol metabolites of individual regioisomers from different types of polyunsaturated fatty acids were easily identified. The limit of detection (LOD) reached as low as 25 nM. The newly developed LC-PCD-DPIS-MS method shows significant advantages in improving the selectivity and therefore can be employed as a powerful tool for profiling vicinal-diol compounds from biological matrices.     

基于磁性颗粒微阵列与双色荧光杂交的单核苷酸多态性分型方法研究

基于磁性颗粒微阵列与双色荧光杂交,建立了单核苷酸多态性(Single nucleoitide polymorphism,SNP)分型方法。将利用不对称扩增得到的含有待检测位点生物素标记的单链PCR产物固定在链亲和素修饰的金磁纳米颗粒(Gold magnetic nanoparticles,GMNPs)表面;将ssDNA-GMNPs混合物点样在底部固定有磁铁的载玻片上构建磁性颗粒微阵列,然后在基因框中与双色荧光探针杂交;杂交完全后,充分洗涤,通过扫描获得分型结果。通过优化不对称PCR的扩增条件,直接扩增出产量较高的单链DNA作为靶序列用于分型。利用本方法对24个样本MTHFR基因的C677T位点多态性进行了检测。实验证明,本方法步骤简单,易实现自动化操作、非常适用于分子诊断与法医鉴定。     

基于质谱分析的代谢组学研究进展

质谱分析技术是代谢组学研究的重要技术之一。该文通过近5年来的文献分析,对基于质谱分析的代谢组学研究方法的新进展,包括样品前处理方法、分析检测方法、数据处理方法等,以及近年来代谢组学在疾病诊断、药物研发、营养学、毒理学、运动医学等领域的应用进展,进行了较全面的综述,并对未来的发展趋势进行了展望。     

Separation and Characterization of the Prostaglandins by Gas Chromatography and Mass Spectrometry

Abstract

Prostaglandins of the A, B, E and F series, together with 8-iso-E₁, have been separated and characterized by gas chromatography and mass spectrometry through the use of TMSi and MO-TMSi derivatives.

These derivatives are suitable for work with GLC systems with flame ionization detectors. Additional derivatives must be sought for detection by electron capture techniques.     

基于液相色谱-串联质谱技术的磷酸化蛋白质组学分析

本文运用稳定同位素双甲基化标记技术、固定相金属离子螯合层析(IMAC)技术并结合液相色谱-串联质谱法研究了仙台病毒感染引起的宿主细胞磷酸化蛋白质组变化。实验发现定量的4 289个磷酸化肽段有~20%的蛋白质磷酸化位点发生了显著变化;通路富集分析表明哺乳动物雷帕毒素靶蛋白(mTOR)通路和剪接体(Spliceosome)通路可能参与宿主细胞对病毒的应答;通过对显著变化磷酸化肽段进行基序分析,发现了9个代表性的磷酸化修饰位点基序。本研究方法对于深入解析抗病毒天然免疫信号通路提供了新线索。