用于蛋白质分析的毛细管等电聚焦-电喷雾质谱接口的改进

蛋白质组学对其分析技术提出了大规模、高通量的要求 [1] .传统的等电聚焦 ( p I) -分子量 ( MW)双向电泳技术 ( 2 D- Gel)尽管在蛋白质组学研究中占有重要地位 ,但其操作繁杂、工作周期长 .Pandey等[1] 将毛细管等电聚焦 ( CIEF)与电喷雾质谱 ( ESIMS)联用 ,使得 p I和 MW两维分离鉴定技术变得简单迅速 .但 CIEF- MS的接口操作需中断高压和将毛细管阴极端插入电喷雾管 ,故引起分析蛋白质的散焦和不重现 .本工作改进了 CIEF- MS接口 ,采用毛细管阴极端和电喷雾针一体化的电喷雾接口 ,无需中断高压 ,实现了 CIEF- MS的在线联…     

鲨鱼软骨血管形成抑制因子的分离纯化及其活性的初步研究

以广东阳江鲨鱼软骨为原料,研究了鲨鱼软骨新生血管形成抑制因子的分离纯化方法,并对其生物学活性进行了初步研究.采用盐酸胍抽提、膜超滤、丙酮分级沉淀、SephadexG-75柱层析和C₄反相高效液相色谱等步骤,分离纯化出一种新的鲨鱼软骨血管生成抑制因子-a(SharkCartilageAngiogenesisInhibitor-a,SCAI-a),分子量为12600,能显著抑制鸡胚绒毛尿囊膜血管的形成.     

17400鲨鱼软骨血管生成抑制因子的纯化及生物学活性研究

采用盐酸胍抽提、膜超滤、丙酮分级沉淀、Sephadex-75柱层析和C₄反相高效液相色谱等分离步骤,从广东阳江鲨鱼软骨中纯化获得新的鲨鱼软骨血管生成抑制因子SCAI-c(SharkCartilageAngiogenesis In-hibitor-c,SCAI-c);SDS-PAGE电泳银染显示为一条带,根据蛋白质的相对迁移率计算,分子量为17400;它对鸡胚绒毛尿囊膜血管的形成具有显著抑制效应,并有明显的浓度依赖关系.SCAI-c与SCAI-a具有类似的生物学特征.     

碘·(N,N'-联吡啶)·(三苯基膦)合铜(Ⅰ)的合成和晶体结构

铜(I)配合物的研究在金属酶的化学模拟和配合物结构及反应性能等研究方面具有重要的理论和实际意义[1].但由于铜(I)配合物不稳定, 且在多数有机溶剂中的溶解度较小,铜(I)配合物的合成比较困难.我们在铜(I)配合物的合成方面积累了一些经验,合成了一系列含有三苯基膦和氮杂环配体的铜(I)配合物[CuX(PPh3)L]n[2～4] (n=1, X=I, L=1,10-phen; n=2, X=Br, I, L=C9H7N),并对它们的结构进行了研究.本文报道一个类似的新配合物[CuI(PPh3)(bpy)](I)的合成和晶体结构, 并把它与其它几个类似的配合物进行了对比.     

血红蛋白与NO分子间相互作用的电化学表征

近年来 ,蛋白质的电化学研究引起了人们广泛兴趣[1～ 5] .利用自组装技术[4 ] 或将蛋白质分子固定于双层磷脂膜、水凝胶、表面活性剂膜及 Al2 O3膜上[6～ 9] 等可成为制备蛋白质膜的有效手段 .血红蛋白(Mr=6 50 0 0 )是生物体内一类重要的蛋白 ,由于对其结构已有较清楚的认识 [10 ] ,因而常被选作探讨生物大分子的电化学行为的模型分子 . NO是一种内皮细胞松弛因子、神经递质和免疫系统的媒介体 ,与生物体内的许多生理过程、疾病的产生与治疗有密切关系 [11～ 13] .因此研究蛋白与 NO之间的相互作用并寻求监测 NO的各种途径具有重要意义…     

荧光L-丙氨酸衍生物的立体选择性合成及对环境状态识别的初步研究

由于终止码抑制技术(nonsense suppressiontechnique)的发展,现在人们已能够将非天然氨基酸定点地(site-specifically)嵌入任何的功能蛋白质中[1-3]。这项技术所带来的一个重要意义是给化学生物学家提供一种潜在、系统、精确地研究蛋白质结构与功能关系的工具[4]。结合具有特殊光学的非天然氨基酸的设计合成与嵌入,科学家们可以利用相关的光谱学手段来探究目标功能蛋白质的内部活性结构,因此,设计和合成具有特殊光谱性质的的非天然氨基酸是合成化学家所感兴趣的课题。A ladan{β-6-′(N-二甲基)氨基-2′-萘甲酰]-L-丙氨酸}的设计合成就是…     

CZE-ESI-MS联用测定小肽混合物的研究

研究肽的分离行为、测定方法及测定条件对蛋白质组学研究具有重要意义 .毛细管电泳 ( CE)作为一种高效、快速的分离方法 ,样品用量少 ,已被广泛应用于生物领域中 ,尤其是小肽和蛋白质的分离分析 .质谱 ( MS)能够进行微量鉴定 ,并提供精确的分子量和结构信息 ,使其成为小肽和蛋白质检测和序列测定的强有力的支撑技术之一 [1～ 3] .其中 ,电喷雾 ( ESI)质谱作为一种软电离技术 ,易与常规的高分辨率分离方法如高效液相色谱、毛细管电泳等实现在线联用 ,具有分离效率高、检测灵敏度高和样品定性方便等特点 ,因而在小肽和蛋白质的测定中得到广…     

La(ClO4)3-BAPOAP-H2O(30 ℃)三元体系的相平衡和Ln2(BAPOAP)3(ClO4)6的合成与表征

安替比林 ( AP)衍生物可用于稀土的分析测定及协同萃取[1] ,安替比林稀土配合物的合成 [2 ] 及其发光性质和磁学性质等 [3,4 ]的研究已有报道 .在对具有芳基侧链的 4- (邻氯苯甲酰基 )氨基安替比林 [5]、4- (对氯苯甲酰基 )氨基安替比林 [6] 及长链桥联的 N ,N -二安替比林 - 1 ,6-己二酰二胺[7,8] 等与稀土盐相互作用进行研究的基础上 ,本文对短链桥联的 N,N -二安替比林乙二酰二胺与镧系高氯酸盐在水溶液中全浓度范围内的相互作用进行了研究 .1　实验部分1 .1　试剂及仪器　L a( Cl O4 ) 3· 8H2 O用 La2 O3( 99.9% )和 1∶ 1高氯…     

采用大分子单体稳定印迹牛血清白蛋白

以牛血清白蛋白为模板蛋白质,聚(丙烯酸羟乙酯-乙烯基咪唑-1-(烯丙基乙酯)-3-乙烯基咪唑氯)[P(HEA-co-VIM-co-[AVIM]Cl)]为大分子功能单体和交联剂,通过氧化还原引发聚合法制备了蛋白质印迹水凝胶.圆二色光谱和同步荧光光谱结果表明,P(HEA-co-VIM-co-[AVIM]Cl)能够较好地维持牛血清白蛋白结构稳定性,而相同质量的HEA,VIM和氯化1-(烯丙基乙酯)-3-乙烯基咪唑([AVIM]Cl)的混合物对牛血清白蛋白的结构破坏严重.选择性吸附和竞争吸附的结果表明,用P(HEA-co-VIM-co-[AVIM]Cl)制备的印迹水凝胶比用HEA,VIM和[AVIM]Cl制备的印迹水凝胶具有更强的选择性和识别能力.在制备过程中维持模板蛋白质结构的稳定性有利于得到具有高识别能力和选择性的印迹聚合物.采用大分子单体印迹蛋白质的方法,可以有效地克服蛋白质在印迹过程中的结构容易变性的缺点,对蛋白质印迹技术的发展具有重要意义.     

AgmC60(OH)n(NO3)m的合成及其热稳定性

自从富勒烯被发现并克量级制备以来,富勒烯独特的结构和性质引起了人们极大兴趣和关注,对富勒烯及其衍生物的研究已迅速发展[1],由于C60具有光诱导剪切DNA、抑制艾滋病毒等特性,因而在生物化学及医学方面具有潜在应用前景,其中水溶性富勒烯衍生物的研究是富勒烯化学较为活跃的研究领域之一[2～5],尤其是羟基化富勒烯(或称富勒醇fullerenols)的合成方向的研究[5]。目前富勒烯羟基衍生物的合成方法已得以深入研究,但其化学反应性能的研究未见报道,本文合成了羟基C60银盐,并对其热稳定性能进行了研究。1　实验部分1 1　主要仪器及试剂C6…     

Identification of Genetic Product — Gm-Csf by Hplc-Esims

ABSTRACT

The genetic product, granulocyte-macrophage colony stimulating-factor (GM-CSF) biosynthesized in this university, is identified through techniques of HPLC and ESIMS in the present study. Results indicate that the experimental molecular weight for the product protein is in agreement with the expected one. Partial sequences are inferred from the tryptic peptide map of product protein. Combining the partial sequence and molecular weights of tryptic peptides, the product protein of GM-CSF has been verified through the Swiss-Prot protein database with satisfactory results.     

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry

Mass spectrometry methods have been used to characterize two proteins: an opioid peptide-containing protein extracted from bovine pituitary, and bovine α-lactalbumin (BAL). A protein that contains β-endorphin was found in bovine pituitary, and that protein was characterized with electrospray ionization mass spectrometry (ESIMS), gel permeation chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), radioimmunoassay, trypsinolysis, and liquid secondary ion mass spectrometry (LSIMS). BAL is a protein that was used as a model to develop analytical methods to study opioid peptide-containing proteins. Commercial BAL was purified by RP-HPLC, and its molecular weight (M.W.) was determined by ESIMS. The shift in mass observed following dithiothreitol (DTT) reduction estimated the number of disulfide bonds. For all of the data obtained for BAL with or without RP-HPLC separation, ESIMS determined the M.W. of the peptides produced by trypsin treatment of BAL, and LSIMS selected a precursor ion, the protonated molecule ion [M + H]⁺, of a tryptic peptide, which was analyzed by tandem mass spectrometry. Following DTT reduction, ESIMS and LSIMS detected each peptide that contained disulfide bonds in that mixture of tryptic peptides.     

微柱高效液相色谱-质谱/质谱快速鉴定混合蛋白质新方法

发展了一种混合蛋白质快速鉴定的新方法.将几种蛋白质的混合物于热变性后直接在溶液中酶解,利用微柱高效液相色谱-离子阱串级质谱进行肽谱/氨基酸序列分析,并结合Mascot数据库搜索处理功能,实现了混合蛋白质快速准确的鉴定.     

Identification of a new gene product of PKIbeta by HPLC-ESIMS

An identification method using high-performance liquid chromatography combined with electrospray mass spectrometry (HPLC-ESIMS) has been developed to verify an expressed gene product of kinase inhibitor (PKIbeta). This protein was expressed in this university for the first time from a newly cloned gene in the cDNA library of human fetal brain. The measured MW (8468.9 Da) of PKIbeta-78 was consistent with expectations. The gene product of PKIbeta-78 was monitored by ESIMS to ensure there was no mis-expressed PKIbeta-70 in the process of gene engineering. The peptide mapping of PKIbeta-78 and its partial sequence were, furthermore, determined. By database searching based on the experimental MWs and partial sequences provided, it was verified that this gene product is a new protein. The pseudosubstrate site and leucine-rich site for the function region of PKIbeta-78 are also confirmed.     

Determination and pharmacokinetics of manidipine in human plasma by HPLC/ESIMS

A sensitive HPLC/ESIMS method was established for the determination of manidipine in human plasma and pharmacokinetics study. After basified plasma with ammonia, manidipine and the internal standard (IS) (felodipine) were extracted with n-hexane and separated on a Hypersil ODS2 column with a mobile phase of methanol-5 mm ammonium acetate solution containing 0.1% acetic acid (85:15, v/v). MS determination was performed by electrospray ionization in the selected ion monitoring mode. Manidipine was monitored at m/z 611.4 and IS at m/z 384. The assay had a calibration range from 0.2 to 20 ng/mL and a lower limit of quantification of 0.1 ng/mL. The method has been successfully applied to the pharmacokinetic study in healthy volunteers.     

Determination of an antipsychotic agent (ICI 204,636) and its 7-hydroxy metabolite in human plasma by high-performance liquid chromatography and gas chromatography-mass spectrometry.

ICI 204,636 (I) is an orally active antipsychotic agent under development for the treatment of schizophrenia in humans. It is partially converted in animals to an active 7-hydroxy metabolite (II). Methods were developed for the simultaneous determination of both analytes in human plasma using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The analytes were extracted from plasma using phenyl solid-phase extraction columns. Quantification by isocratic HPLC was performed in the reversed-phase mode with detection at 250 nm. Extracts were derivatized to trimethylsilyl ethers for quantification by GC-MS using selected-ion monitoring. Both assays were evaluated for consistency of response, precision, accuracy and specificity. Limits of quantification for I and II by HPLC were 15 and 20 ng/ml, respectively; limits of quantification for I and II by GC-MS were 2 and 5 ng/ml, respectively. Both methods were applied to the analysis of clinical samples from oral dosing studies with I.     

Electrospray ionization mass spectrometry of biotin binding to streptavidin

Stepwise binding of biotin to streptavidin via several intermediates was monitored with electrospray ionization mass spectrometry (ESIMS). Protein ligand interactions that result in conformational changes could be recognized with ESIMS by a mass shift and a change of the average multiple charge state of this protein. In addition, mass spectrometry for the ions in the gas phase revealed a much greater strength of the noncovalent bonds between the streptavidin subunits in the tetrameric complex than between the streptavidin and biotin molecules and remarkable differences in stability for the different charge states of the biotin-streptavidin noncovalent complex.     

High-performance liquid chromatographic determination of loracarbef, a potential metabolite, cefaclor and cephalexin in human plasma, serum and urine

A high-performance liquid chromatographic (HPLC) method is reported for the determination of a new carbacephem antibiotic, loracarbef, a hydroxylated analogue, and two cephalosporins, cefaclor and cephalexin, in plasma, serum, and urine. The antibiotics are extracted from plasma by means of C18 solid-phase cartridges. Urine samples are diluted with water and directly injected on the HPLC system. The HPLC system utilizes a Supelcosil LC-18-DB (250 mm x 4.6 mm I.D.) reversed-phase column and ultraviolet detection at 265 nm. The limit of quantitation is 0.5 micrograms/ml for each compound. Excellent correlation of plasma concentrations is shown between results determined by HPLC and those obtained by microbiological agar-well diffusion assays. Stability studies of loracarbef in human plasma show the antibiotic to be stable for at least 24 h at room temperature and for at least twelve months at -20 degrees C.     

Liquid chromatography/electrospray ionization mass spectrometry method for the determination of the active metabolite M-1 of suplatast tosilate in human plasma

A liquid chromatography/electrospray ionization mass spectrometry (LC/ESIMS) method for the determination of 4-(3-ethoxy-2-hydroxypropoxy) acrylanilide (M-1), the active metabolite of suplatast tosilate, in human plasma was established. Plasma samples were extracted with diethyl ether, separated on a C(18) column with a mobile phase of acetonitrile-10 mm ammonium acetate solution containing 0.1% formic acid (28:72, v/v) and detected by ESIMS. The method was linear over the concentration range 0.15-60.0 ng/mL. The lowest limit of quantification was 0.15 ng/mL. The intra- and inter-run relative standard deviations obtained from three validation runs were all less than 8.6%, and the intra- and inter-run relative errors were all less than 3.1%. The method was successfully applied for the evaluation of pharmacokinetic profiles of M-1 in healthy volunteers.     

Non－decoloured In－gel Digestion of Coomassie Blue－stained Proteins

A simplified method is presented for tryptic digestion of Coomassie brilliant blue(CBB)-stained proteins in polyacrylamide gels. Compared with conventional methods, the proposed method does not require a re-moval of the dye before digestion, and is thus faster and saves a lot of labor. The resulted digest canbe ana-lyzed by either RPLC/ESIMS or MALDI MS for identification of the protein in a conventional way. Model studies with bovine serum albumin(BSA) showed that 50 ng of the protein could be routinely identified. The simplified procedure displays a tendency to produce more incompletely cleaved peptides, which is favorable for improving the sequence coverage.