不稳定性心绞痛血瘀证的血浆蛋白质组学研究

为了寻找冠心病不稳定性心绞痛血瘀证血浆差异表达蛋白, 探索冠心病不稳定性心绞痛血瘀证的蛋白质组学特点. 采用差异凝胶双向电泳和质谱联用技术对12例冠心病不稳定性心绞痛血瘀证患者和12例健康人血浆进行比较研究. 初步发现了Fibrinogen β chain, Fibrinogen γ chain, α1-Antitrypsin, Haptoglobin β chain, Haptoglobin α2 chain在冠心病不稳定性心绞痛血瘀证患者中高表达, ApoA-IV, ApoA-I, Transthyretin, ApoJ在冠心病不稳定性心绞痛血瘀证患者中低表达. 差异表达蛋白根据功能可分为以下三类: (1)急性时相反应负相蛋白; (2)载脂蛋白; (3)凝血相关蛋白. 冠心病不稳定性心绞痛血瘀证可能与炎症反应、脂代谢紊乱以及凝血功能异常相关.     

凝胶电泳-毛细管液相色谱-质谱联用技术分析大肠癌肿瘤与癌旁组织的差异蛋白质组

通过凝胶电泳将大肠癌患者的手术标本组织(肿瘤与癌旁)中的总蛋白按照分子量分成不同的组分,经胶内水解后,利用毛细管液相色谱-质谱联用技术对各个组分的多肽混合物进行蛋白质组学分析,共鉴定出29种差异表达的蛋白质,其中肿瘤组织中表达上调的蛋白质19种,如Fibrinogen beta chain,Vimentin,Fi-brinogen gamma chain,Histone H2A type 1-B/E,Isoform 1 of Periostin,Fibrinogen alpha chain,Histone H3.1,Al-pha-enolase,Protein disulfide-isomerase A3等;在肿瘤组织中表达下调的有10种蛋白质,如Sarcomeric tropomy-osin kappa,Transgelin,Heat shock protein beta-1,Talin-1,Isoform 2 of Vinculin,Isoform 1 of Filamin-C等.通过蛋白质印迹实验对其中的踝蛋白1(Talin-1)和烯醇化酶(Alpha-enolase)的蛋白质组学鉴定结果进行了验证.生物信息学分析表明在大肠癌组织中表达上调的蛋白多具有胞外分泌的属性,提示这些蛋白很有可能会渗透到机体内的循环系统中,从而在患者血液或者腹水中能够被检测到;而在大肠癌组织中表达下调的蛋白则多为细胞骨架、胞外基质纤维等的组成成分,这些蛋白的下调常与肿瘤细胞向周围组织的侵袭和转移有密切关系.本研究为大肠癌的早期诊断、大肠癌初筛等方面的研究等提供了基础数据,对进一步深入研究大肠癌发病机制具有重要意义.     

维拉帕米作用下急性心梗大鼠比较蛋白质组学研究

以急性心梗大鼠为研究对象, 应用双向凝胶电泳法(2-DE)分析比较了维拉帕米作用下急性心梗大鼠心肌蛋白表达的差异, 从蛋白质水平探讨了维拉帕米心肌保护作用的发生机制. 结果表明, 与假手术组及模型组相比, 维拉帕米给药组心肌组织中有8个蛋白点表达显著上调, 7个蛋白点表达显著下调. 采用质谱(MALDI-TOF-MS)分析结合数据库检索, 共鉴定了其中的15种蛋白质, 可按功能分为如下4类: (1) 能量代谢及线粒体功能相关蛋白; (2) 氧化应激相关蛋白; (3) 细胞骨架蛋白; (4) 其它蛋白. 研究结果表明, 维拉帕米的心肌保护作用与恢复心肌损伤过程中的能量供应及对抗氧化应激等作用有关.     

蛋白质组学技术筛选与鉴定癫痫疾病的差异蛋白质

应用蛋白质组学双向凝胶电泳(Two-dimensional gel electrophoresis, 2DE)和质谱技术, 定量分析和鉴定了癫痫组(n=3)和正常组(n=3)脑组织的差异表达蛋白, 以从蛋白质水平上揭示癫痫病的发机制. 结果表明, 凝胶图谱可辨识2500~3000个蛋白点, 对21个显著差异表达蛋白点进行质谱鉴定和SwissProt数据库检索, 得到17个癫痫差异蛋白, 其中2个蛋白在癫痫组织中表达上调, 15个蛋白表达下调. 部分蛋白与癫痫的关系属首次报道. 这些蛋白与癫痫的发生发展相关, 可能成为癫痫的分子标志物和药物治疗的靶向蛋白.     

恐惧记忆相关蛋白的蛋白质组学研究

应用双向凝胶电泳结合质谱鉴定和数据库检索, 分析比较了CD1和C57BL/6J小鼠经条件性恐惧实验后海马蛋白表达的差异, 探讨了与恐惧记忆相关的蛋白质. CD1和C57BL/6J小鼠经条件性恐惧实验后, 海马蛋白表达存在明显差异, 29种蛋白(31个蛋白点)与恐惧记忆的形成显著相关. 其中24个蛋白点表达显著上调, 7个蛋白点显著下调. 与恐惧记忆相关的蛋白按功能可分为如下6类: (1) 能量代谢或线粒体功能相关蛋白; (2) 神经发育相关蛋白; (3) 信号转导相关蛋白; (4) 细胞骨架相关蛋白; (5) 氨基酸代谢和蛋白分解相关蛋白; (6) 伴侣蛋白. 这些恐惧记忆形成的相关蛋白深化了对恐惧记忆脑机制的认识, 为研究和治疗认知相关疾病提供了新靶标.     

空间记忆相关蛋白的蛋白质组学研究

C57BL/6J小鼠在评价空间记忆的多T迷宫(MTM)和Barnes迷宫(BM)中表现不同,本研究拟寻找导致这种行为差异的海马蛋白.应用双向凝胶电泳结合质谱鉴定和数据库检索,分析比较经两种迷宫训练测试后小鼠海马蛋白水平的不同,发现经过BM和MTM训练的小鼠有29种蛋白表达存在明显差异.其中,在BM训练组中5种蛋白表达上调,而在MTM组中24种蛋白表达上调.与空间记忆相关的蛋白按功能可分为如下6类:(1)能量代谢相关蛋白;(2)细胞骨架相关蛋白;(3)分子伴侣;(4)神经发育相关蛋白;(5)转录因子和蛋白合成相关蛋白;(6)信号转导蛋白.本研究结果丰富了空间记忆的机制,对于神经科学的进一步发展具有启发意义.     

快速老化模型小鼠血清蛋白质组学分析比较

以6月及12月龄SAMP 8及同龄SAMR 1为研究对象, 应用双向凝胶电泳法, 分析比较了快速老化模型小鼠(Senescence accelerated mice, SAM)的快速老化亚系SAMP 8及抗快速老化亚系SAMR 1血清蛋白表达的差异. 与同龄SAMR 1比较, 6月龄SAMP 8血清中有15个蛋白点表达显著上调, 3个蛋白点表达显著下调, 7个蛋白点只在SAMP 8中有表达; 12月龄SAMP 8血清中有9个蛋白点表达显著上调, 7个蛋白点表达显著下调, 12个蛋白点只在SAMP 8中有表达. 应用质谱进行肽质量指纹图谱分析和数据库检索共鉴定了19种蛋白质. 其中6个蛋白只在6月龄SAMP 8中表达, 4个蛋白只在12月龄SAMP 8中表达. 此外, 在6月龄及12月龄SAMP 8血清差异蛋白中, 存在9个共同的差异蛋白, 按照功能可分为4类: (1) 免疫相关蛋白; (2) 老化相关蛋白; (3) 糖代谢及神经元凋亡相关蛋白; (4) 其它蛋白. 上述研究结果显示, SAMP 8和SAMR 1血清蛋白表达存在明显差异, 其中一些差异蛋白可能是SAMP 8老化进程中相关病理生理变化的重要原因.     

梅花鹿茸快速生长及骨化期相关差异表达蛋白分析

采用同位素标记相对定量(iTRAQ)技术、超高效液相色谱-质谱联用技术和生物信息学分析方法对东北梅花鹿快速生长期(60 d)与骨化期鹿茸(90 d)进行比较蛋白质组学研究,共鉴定出127种差异蛋白。与骨化期鹿茸比较,快速生长期鹿茸中显著上调的差异蛋白80种,显著下调差异蛋白47种,这些差异表达蛋白主要分布在细胞外基质、核小体、珠蛋白-血红蛋白复合物、肌动蛋白丝、内质网-高尔基中隔室、内质网官腔及核内膜等部位。快速生长期鹿茸显著上调的差异蛋白主要是参与血氧运输、神经生长和再生、软骨和骨组织发育及ATP合成的功能蛋白,而显著下调的差异蛋白主要为参与软骨内骨化过程的功能蛋白,这些蛋白在鹿茸不同生长时期表达量的变化与鹿茸的快速生长和骨化进程密切相关。本研究为系统揭示鹿茸快速生长及骨化的分子机制提供了基础数据,同时对研究鹿茸的药效物质基础及临床应用具有重要的指导意义。     

血管外膜肌成纤维细胞分化相关蛋白研究

为寻找涉及血管紧张素II (AngII)和转化生长因子β1 (TGF-β1)诱导的血管肌成纤维细胞(MF)分化的蛋白, 本研究采用双向电泳和质谱从整体水平检测了MF分化前后蛋白表达谱的变化, 共找到41个差异表达的蛋白点, 表达水平和/或蛋白位置在Ang II和TGF-β1刺激后都发生明显变化的蛋白点14个, 4个蛋白上调, 6个蛋白下调, 2个蛋白位置发生明显变化, 2个蛋白表达上调,位置也发生变化; 只在Ang II诱导的MF中表达发生变化的蛋白20个, 只在TGF-β1诱导的MF中表达发生变化的蛋白7个. 选取Ang II和TGF-β1共同调节的14个蛋白进行质谱鉴定, 结果除骨架蛋白外, 首次发现MF分化同泛素蛋白酶体系统和嘌呤合成有关; septin 2的下调可能是成纤维细胞分化的标志. 本研究运用蛋白质组学技术发现了新的参与MF分化的蛋白质, 为进一步研究和干预细胞表型转化提供了新的思路和靶点.     

快速老化模型小鼠海马蛋白质组学初步研究

应用双向凝胶电泳结合质谱鉴定, 分析比较6月龄和12月龄快速老化模型小鼠(Senescence-accele-rated mouse, SAM)的快速老化亚系SAM-prone/8(SAMP8)及抗快速老化亚系SAM-resistance/1(SAMR1)海马蛋白表达的差异, 从蛋白质水平初步探讨与老化相关的学习记忆功能障碍的发生机制. 结果表明, 与同龄SAMR1比较, 6月龄SAMP8海马中有15个蛋白点表达显著上调, 5个蛋白点表达显著下调; 12月龄SAMP8海马中有12个蛋白点表达显著上调, 2个蛋白点表达显著下调, 2个蛋白点只在SAMP8中有表达. 应用质谱分析结合数据库检索, 共鉴定了22种蛋白质. 6月龄和12月龄SAMP8与SAMR1海马中表达有明显变化的蛋白按功能可分为如下4类: (1) 能量代谢相关蛋白; (2) 线粒体功能相关蛋白; (3) 信号转导相关蛋白; (4) 其它蛋白. 研究结果表明, SAMP8和SAMR1海马蛋白表达存在明显差异, 其中一些蛋白与SAMP8随龄出现的学习记忆功能减退相关, 并可能为研究或发现促智药物作用的新蛋白靶标提供线索.     

High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.     

Composition and genetic variability of proteins from nuclear fractions of mouse (DBA/2J and C57BL/6J) liver and brain

Proteins from nuclear plasma of mouse liver and brain and from the nuclear membranes of mouse liver were separated by two-dimensional electrophoresis. For the purpose of comparison, liver cytosol proteins were also investigated. The protein samples were prepared from two inbred strains of the mouse (DBA/2J, C57BL/6J) and their hybrids. The patterns obtained were compared with regard to the composition and genetic variability (qualitative and quantitative variants) of proteins from different nuclear fractions and organs. The percentage (greater than 30%) of spots common to different organs (liver, brain), but from the same nuclear fraction (plasma) was greater than the percentage (less than 20%) of spots common to different cell and nuclear fractions (cytosol, nuclear plasma and nuclear membranes) of the same organ (liver). Quantitative genetic variants occurred much more frequently than qualitative genetic variants (5.1% vs. 0.2%; liver nuclear plasma). The incidence of genetic variants was much higher in liver (5.3%) than in brains (0.0%), and higher in solubilized nuclear proteins (5.3%) than in structure-bound nuclear proteins (2.1%).     

A sensitive method for determination of salvianolic acid A in rat plasma using liquid chromatography/tandem mass spectrometry

Salvianolic acid A (SAA), a major effective constituent of Salvia miltiorrhizas, is widely used in traditional Chinese medicine. A sensitive rapid analytical method was established and validated for SAA in rat plasma, which was further applied to assess the pharmacokinetics of SAA in rats receiving a single oral dose of SAA. The method used liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode with chloramphenicol as the internal standard. A simple liquid-liquid extraction based on ethyl acetate was employed. The combination of a simple sample cleanup and short chromatographic run time (3 min) increased the throughput of the method substantially. The method was validated over the range 1.4-1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1.4 ng/mL for SAA in plasma. Intra- and inter-day accuracies for SAA were 95-113 and 98-107%, and the inter-day precision was less than 12%. This method is more sensitive and faster than previous methods. After a single oral dose of 100 mg/kg of SAA, the mean peak plasma concentration (Cmax) of SAA was 318 ng/mL at 0.5 h, the area under the plasma concentration-time curve (AUC0-12 h) was 698 +/- 129 ng.h/mL, and the elimination half-life (T1/2) was 3.29 h.     

Validation of a high-performance chromatographic method for determination of cefotaxime in biological samples

An analytical method for detecting and quantifying cefotaxime in plasma and several tissues is described. The method was developed and validated using plasma and tissues of rats. The samples were analyzed by reversed phase liquid chromatography (HPLC) with UV detection (254 nm). Calibration graphs showed a linear correlation (r > 0.999) over the concentration ranges of 0.5–200 μg/mL and 1.25–25 μg/g for plasma and tissues, respectively. The recovery of cefotaxime from plasma standards prepared at the concentrations of 25 μg/mL and 100 μg/mL was 98.5 ± 3.5% and 101.8 ± 2.2%, respectively. The recovery of cefotaxime from tissue standards of liver, fat and muscle, prepared at the concentration of 10 μg/g was: 89.8 ± 1.2% (liver), 103.9 ± 6.5% (fat) and 97.8 ± 2.1% (muscle). The detection (LOD) and quantitation (LOQ) limits for plasma samples were established at 0.11 μg/mL and 0.49 μg/mL, respectively. The values of these limits for tissues samples were approximately 2.5 times higher: 0.3 μg/g (LOD) and 1.25 μg/g (LOQ). For plasma samples, the deviation of the observed concentration from the nominal concentration was less than 5% and the coefficient of variation for within-day and between-day assays was less than 6% and 12%, respectively. The method was used in a pharmacokinetic study of cefotaxime in the rat and the mean values of the pharmacokinetic parameters are given.     

High-performance liquid chromatographic assay for mitoxantrone in plasma using electrochemical detection

A sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of mitoxantrone in plasma using electrochemical detection. Bisantrene was chosen as the internal standard. A reversed-phase, 10-microns muBondapak C18 analytical column (30 cm X 3.9 mm) with an isocratic mobile phase of 28% acetonitrile in 80 mM sodium formate buffer (pH 3.0) was used. The eluent was monitored by both electrochemical detection at an applied potential of +0.75 V vs. Ag/AgCl and visible absorbance at 660 nm. Only electrochemical detection was able to quantitate the internal standard and provided ten times higher sensitivity than visible absorbance for mitoxantrone with a detection limit as low as 0.1 ng/ml. Calibration curves in the range 0.1-1000 ng/ml showed good linearity (r = 0.998) and precision (coefficient of variation less than 10%). This HPLC method utilized a reproducible and inexpensive liquid-liquid extraction procedure. Using methylene chloride, the extraction efficacy of mitoxantrone from plasma was 85.3% with a coefficient of variation less than 2.1%. This new assay was then applied to measure mitoxantrone concentrations in plasma obtained from two leukemic patients receiving 12 mg/m2 mitoxantrone as a 1-h infusion.     

Quantification of malondialdehyde by HPLC‐FL – application to various biological samples

Malondialdehyde (MDA) is stabile product of lipid peroxidation (LPO), and therefore MDA is frequently used as a biomarker of LPO. To determine MDA level in various biological samples (human plasma, fish liver tissue and cells in culture), we used an HPLC method with fluorescent detection based on 2‐thiobarbituric acid (TBA) assay. The method was validated by the use of spiked pooled plasma samples. In tested concentration range (0.15–3.0 µmol/L) the method was linear (R² = 0.9963), the between‐day variability (coefficient of variations, CVs) was between 4.7 and 7.6%, the within‐day variability CVs was between 2.6 and 6.4% and recovery was between 91.2 and 107.6%. The level of MDA in human plasma (healthy male, non‐smokers, 46.3 ± 4.7 years; N = 38) was 2.2 ± 1.4 µmol/L; that in liver tissue of common carp (Cyprinus carpio; N = 12) was 0.02 ± 0.004 µmol/g tissue, and in cultured cells (human laryngeal carcinoma cells; N = 10) it was 0.18 ± 0.02 nmol/mg proteins. The HPLC‐FL method is rapid, accurate and reliable to follow the extent of LPO in various biological samples, particularly in samples in which a low level of MDA is expected, such as cells in culture. Owing to the rapid analytical process and run time, it can be used for routine analysis of MDA in clinical laboratory. Copyright © 2014 John Wiley & Sons, Ltd.     

Serum and plasma beta-carotene levels measured with an improved method of high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method specifically developed to allow simple and rapid determination of beta-carotene concentrations in serum and plasma is reported. Using a method modified from a previously published technique, serum and plasma proteins are denatured by exposure to perchloric acid, and beta-carotene is subsequently extracted into an organic matrix consisting of ethyl acetate-tetrahydrofuran (1:1); no evaporation step is required. Separation is achieved using isocratic elution from a reversed-phase C18 column with UV detection at 436 nm. Recovery of beta-carotene from water and plasma was greater than 98.1%; beta-carotene was stable in the extraction matrix for at least 4 h. Three anticoagulants (oxalate, citrate, and EDTA) caused losses of beta-carotene; perchloric acid and tetrahydrofuran could also destroy beta-carotene under certain conditions. Each run required less than 15 min; within-day coefficient of variation for identical samples averaged 2.3%, between-day coefficient of variation was 4.4% and sensitivity was better than 10 ng/ml. Stability of beta-carotene in plasma was also examined. This method permits a simple, rapid, sensitive, precise, and accurate determination of beta-carotene using 0.5 ml of serum or heparinized plasma.     

Stereospecific high-performance liquid chromatographic determination of tocainide

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.     

Validation of a high-performance chromatographic method for determination of cefotaxime in biological samples

An analytical method for detecting and quantifying cefotaxime in plasma and several tissues is described. The method was developed and validated using plasma and tissues of rats. The samples were analyzed by reversed phase liquid chromatography (HPLC) with UV detection (254 nm). Calibration graphs showed a linear correlation (r > 0.999) over the concentration ranges of 0.5–200 μg/mL and 1.25–25 μg/g for plasma and tissues, respectively. The recovery of cefotaxime from plasma standards prepared at the concentrations of 25 μg/mL and 100 μg/mL was 98.5 ± 3.5% and 101.8 ± 2.2%, respectively. The recovery of cefotaxime from tissue standards of liver, fat and muscle, prepared at the concentration of 10 μg/g was: 89.8 ± 1.2% (liver), 103.9 ± 6.5% (fat) and 97.8 ± 2.1% (muscle). The detection (LOD) and quantitation (LOQ) limits for plasma samples were established at 0.11 μg/mL and 0.49 μg/mL, respectively. The values of these limits for tissues samples were approximately 2.5 times higher: 0.3 μg/g (LOD) and 1.25 μg/g (LOQ). For plasma samples, the deviation of the observed concentration from the nominal concentration was less than 5% and the coefficient of variation for within-day and between-day assays was less than 6% and 12%, respectively. The method was used in a pharmacokinetic study of cefotaxime in the rat and the mean values of the pharmacokinetic parameters are given. Received: 25 May 1998 / Revised: 27 July 1998 / Accepted: 1 August 1998     

高效液相色谱法测定非离子造影剂欧乃帕克

建立了一种快速、灵敏的细径柱高效液相色谱测定非离子造影剂欧乃帕克的方法。用高氯酸去除血浆蛋白,流动相采用简单的7%（体积分数）甲醇水溶液体系。方法的线性范围为0.5~500 mg/L ,相关系数大于 0.999 8 。欧乃帕克的最低检测量为154 pg,保留时间为(3.46 ± 0.02) min,总分析时间少于5 min。日内、日间相对标准偏差(RSD)分别小于4.3%和11.4%。方法的平均回收率大于91.51%。