用阳极溶出伏安法测定铅血清蛋白络合物、条件生成常数及血清蛋白的分子量

本文提出利用阳极溶出伏安法测定铅离子与人血清白蛋白,人血清丙种球蛋白和牛血清白蛋白所生成的金属蛋白络合物的组成及其条件生成常数,并测定与铅离子相结合的蛋白质的分子量.     

天花粉蛋白的C端测定

天花粉蛋白(trichosanthin)是从葫芦科植物(Culurbitaceae)括楼(trichosantheskerilowii Maxim)的块根中提取的一种植物性蛋白质。该蛋白质有中期妊娠引产、抗早孕和治疗其它有关妇产科疾病的效力。根据已经测定的结果,该蛋白质系由19种约219个氨基酸组成,估计其分子量为24000。本项研究的主要目的是测定天花粉蛋白的C端和C端的部分氨基酸顺序。     

血清多肽组及其个体差异的纳升液相色谱-高分辨串联质谱分析

分析和比较疾病组及健康对照组的混合样品是血清多肽组生物标记物研究的常用方法,但对健康个体多肽组的差异和共性关注较少.本研究利用纳升液相色谱-高分辨四级杆飞行时间质谱鉴定健康人混合血清样品(20例)的多肽组,阐明血清多肽组的分子量分布等一般特征,进而选取6例个体样品单独分析并与混合样品的分析结果进行比较,说明正常健康样品之间的个体差异和共同成分.结果表明,可鉴定序列的血清多肽组的分子量范围在7000 Da以下,纤维蛋白原α链等蛋白质所属肽段的检出频率最高,肽段在蛋白质水平上分布具有不均一性,排在前10％的蛋白质占据了约50％的总肽段,而后40％的蛋白质只有1条检出肽段.此外,在所有样品中都检测到了来自于8个蛋白质的12个共同肽段,检测到了N端乙酰化、氨基酸氧化、磷酸化、脱氨化和脱水等翻译后修饰和明显的阶梯序列现象.本研究在肽段序列水平分析了血清多肽组的基本特征和个体差异,可为血清多肽组生物标志物研究提供参考.     

AEOT/异辛烷/水反胶团体系对血红蛋白的提取

自Riet等报道以反胶团技术提取蛋白质以来,有关反胶团技术提取生物活性物质的报道较多,但研究对象多为分子量较小的蛋白质及酶.由表面活性剂丁二酸二(2-乙基己基)酯磺酸钠(AOT)形成的反胶团体系水含量W₀,有机相中水的摩尔浓度与表面活性剂摩尔浓度之比)较小,提取率偏低.     

分子量条带用蛋白质

蛋白质的生物化学、生物物理学及化学生物学研究中,凝胶电泳是常见且重要的分析手段。然而其背后所蕴含的一众有关蛋白质结构及功能的知识点长期被国内外本科相关专业教学忽略。从探讨蛋白质凝胶电泳的分子量指示物的筛选标准出发,分析比较了几类常见的分子量指示物,并基于一级结构和高级结构的稳定性对分子量条带用蛋白质的筛选提出了一些合理的推论。     

改性介孔材料制备及其对低分子量蛋白质的在线选择富集研究

制备了3种内外表面修饰不同官能团的硅基介孔材料,采用傅立叶变换红外光谱对修饰后材料的官能团进行了表征,并采用高效液相色谱对材料选择性富集低分子量蛋白质的能力进行了考查。3种材料对低分子量蛋白质和小肽均具有选择性富集能力,其中乙烯-二醇材料的富集能力最好。采用乙烯-二醇介孔材料装填预柱,考察了其对低分子量蛋白质的在线富集能力。高分子量蛋白质(卵清蛋白,分子量43000Da)在该预柱上没有保留,在死时间里直接流出柱外,而低分子量蛋白质(胰岛素,分子量5700Da)在该预柱上得到了较好的选择性萃取。保留在柱上的低分子量样品通过提高流动相中的有机相含量进行洗脱。研究工作对低分子量蛋白质和小肽的在线富集提供了一种新技术。     

蚕蛹水解液的分子量测定

凝胶过滤法是测定蛋白质分子量的常用方法之一,但标准蛋白质不易得到,且保存较困难。本工作用已知分子量的聚乙二醇作标样,采用凝胶过滤色谱外插法定量测定蚕蛹酶解液的分子量。实验色谱柱填料用Sephadex—50,聚乙二醇分子量为20000～1000,洗脱液用紫外光谱检测。根据洗脱体积与蛋白质分子量成直线关系的原理,作分子量与洗脱体积的关系曲线,并用已知分子量的细胞色素C验证。实验表明,该法所得结果与聚丙烯酰胺凝胶电泳和GPC法的测定结果一致。     

尿毒症毒素多肽二级结构的测定和预测

尿毒症患者由于肾脏损伤,使某些代谢物质积蓄在体内而引起中毒,最终导致死亡,由于其分离纯化过程复杂,至今人们对尿毒症中分子积累物的认识仍很有限。目前,已确定的中分子毒物以蛋白质和多肽为主,在前期的工作中,我们从尿毒症患者血清中分离出分子量为1007的中分子物质,实验证明该物质可加速肾衰兔的死亡。     

流动注射光度法测定黄酒中蛋白质的含量和分子量分布

将流动注射技术引入Bradford蛋白质测定法,利用Sepadex G-100凝胶层析柱分离黄酒中的蛋白质,可直接测定蛋白质的含量和分子量分布。成品酒（熟酒）为5．32g/L（6．86g／L）;高分子蛋白质（Fw〉25000）占10．52％（13、08％）;中分子蛋白质（FW：25000～5000）占49、12％（54．12％）;小分子蛋白质（FW〈5000）占40、36％（32、80％）,并得到黄酒中蛋白质含量与分子量分布的曲线谱图,可监控和预测黄酒的品质。     

胸腔积液中低分子量蛋白质的提取

胸腔积液的生化指标已经成为临床疾病诊断的重要依据.引起胸腔积液的疾病中,人们最为关注的是恶性肿瘤引起的胸水渗漏(恶性胸水),并且做了比较系统的研究~([1,2]).在胸腔积液中寻找蛋白模式的肿瘤标志物越米越得到人们的重视.与血清相似,胸腔积液中白蛋白也干扰其它低丰度蛋白的检测.本研究建立一种简单的圆盘电泳方法,提取胸腔积液中低分子量蚩白,去除高分子量蛋白,包括人血白蛋白.     

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.     

Proteomic analysis of microvesicles in human saliva by gel electrophoresis with liquid chromatography-mass spectrometry

Microvesicles (MVs) have been shown to affect the physiology of neighboring recipient cells in various ways. They play an important role in tumor progression/metastasis and angiogenesis in cancer and may be useful therapeutic tools, as well as a mechanism of cell-to-cell communication. They have been visioned as an important biomarker or biomarker source for the detection of different diseases. Human saliva is a biological fluid with enormous diagnostic potential, which harbors plenty of salivary MVs. The goal of this study is to investigate the proteomic profiling of MVs in human saliva through a simple preparation procedure by using filtration and centrifugation. Gel electrophoresis was combined with LC–MS/MS (liquid chromatography–mass spectrometry) for the proteomic analysis of MVs. After SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) protein separation, the whole lane was cut into 25 bands, and each band was subjected to in-gel trypsin digestion. The peptides extracted from each band were loaded to LC–MS/MS for protein identification. Through protein database search, 63 proteins were identified for human salivary MVs. Several members of different protein families were identified, including annexin, keratin, actin, immunoglobulin and S100. This study showed that although there was an overlap with the proteins from human saliva and salivary exosomes, salivary MVs contained their own unique proteins. These results will poise human salivary MVs as a non-invasive tool for the early detection of different diseases.     

Combination of FASP and fully automated 2D‐LC‐MS/MS allows in‐depth proteomic characterization of mouse zymogen granules

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two‐dimensional gel electrophoresis or one‐dimensional SDS–PAGE, followed by in‐gel tryptic digestion. In order to overcome the disadvantage of in‐gel digestion and to carry out further in‐depth proteomic analysis of the zymogen granules, in this study, by combining a filter‐aided sample preparation method and fully automated 2D‐LC‐MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions. Copyright © 2012 John Wiley & Sons, Ltd.     

非小细胞肺癌患者尿中Exosomes蛋白质组的差异表达分析

除血浆之外,尿液是另一种寻找潜在生物标志物的重要生物材料。本研究以200000×g超速离心法分离正常人和非小细胞肺癌(NSCLC)患者尿液中的Exosomes,运用1DSDS-PAGE对Exosomes蛋白质组进行分组,从电泳胶上切取正常组和疾病组的20～31kDa条带,胰蛋白酶酶解后,进行HPLC-CHIP-MS/MS分析,并通过UniProtKB/SWISS-PORT数据库搜索鉴定了24种蛋白质,其中在NSCLC患者尿液Exosomes蛋白质组中发现了8种差异表达蛋白,包括免疫球蛋白κ的3个片段、2种Ras相关蛋白、谷胱甘肽S转移酶A2、血清淀粉样P成分前体和磷脂酰乙醇胺结合蛋白1。     

Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.     

Through the eye of an electrospray needle: mass spectrometric identification of the major peptides and proteins in the milk of the one‐humped camel (Camelus dromedarius)

The milk of the one‐humped camel (Camelus dromedarius) reportedly offers medicinal benefits, perhaps because of its unique bioactive components. Milk proteins were determined by (1) two‐dimensional gel electrophoresis and peptide mass mapping and (2) liquid chromatography–tandem mass spectrometry (LC–MS/MS) following one‐dimensional polyacrylamide gel electrophoresis. Over 200 proteins were identified: some known camel proteins including heavy‐chain immunoglobulins and others exhibiting regions of exact homology with proteins from other species. Indigenous peptides were also identified following isolation and concentration by two strategies: (1) gel‐eluted liquid fraction entrapment electrophoresis and (2) small‐scale electrophoretic separation. Extracts were analyzed by LC–MS/MS and peptides identified by matching strategies, by de novo sequencing and by applying a sequence tag tool requiring similarity to the proposed sequence, but not an exact match. A plethora of protein cleavage products including some novel peptides were characterized. These studies demonstrate that camel milk is a rich source of peptides, some of which may serve as nutraceuticals. Copyright © 2013 John Wiley & Sons, Ltd.     

The resistance of metallothionein to proteolytic digestion: an LC-MS/MS analysis

Metallothioneins (MTs) are a family of cysteine-rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC-MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal-saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture.     

基于M13噬菌体展示单链抗体文库的新型蛋白质均衡器的研制及其在肾病患者尿蛋白分析中的应用

通过将展示单链抗体(scFv)文库的M13噬菌体固定于冷凝胶整体柱表面制备了一种新型蛋白质均衡器。由于展示的scFv片段具有种类多、数目大以及与蛋白质结合的特异性强等优点,因此其非常适合用于复杂蛋白质样品的预处理。将肾病患者尿蛋白在平衡器上反复上样5次后,依次使用2 mol/L NaCl、50 mmol/L Gly-HCl(pH 2.5)和凝血酶溶液洗脱与均衡器结合的蛋白质,收集各馏分,并采用串联微柱反相液相色谱-电喷雾质谱进行蛋白质的分离鉴定。与未经均衡器处理的样品相比,鉴定的蛋白质数目由142个提高到396个。此外,凝胶电泳的分析结果显示,在上样流出液中蛋白质的浓度差异明显变小,大量的高丰度蛋白质存在于NaCl洗脱液中。上述结果表明,基于M13噬菌体展示scFv文库的新型蛋白质均衡器能够有效减小样品中蛋白质的浓度差异,有利于发现更多的低丰度蛋白质。     

Analysis of expression and comparative profile of normal placental tissue proteins and those in preeclampsia patients using proteomic approaches

Preeclampsia (PE) is a complex and serious condition of pregnancy. Trophoblasts in human placenta can be separated and collected by laser capture micro-dissection (LCM). Protein in trophoblasts have been extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), finally 962 unique proteins are identified by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Comparison of differential expressed proteins in normal and those in PE are investigated. Two-dimensional electrophoresis (2DE) and MS were used to identify differential expressed proteins. 13 differential expressed proteins include signal transduction protein, molecular chaperone, cell skeleton proteins are identified, in which 3 proteins are down-regulated and 10 proteins are up-regulated. They might be correlated with the cause of PE.     

多维离子交换色谱分离和串联质谱鉴定在小鼠肝脏膜蛋白质组分析中的应用

膜蛋白质在变性剂作用下能够较充分地溶解。根据这一特点,我们试图在变性剂溶液中采用串联离子交换色谱法分离小鼠肝脏膜蛋白质。将小鼠肝脏膜蛋白质溶解于含有4 mol/L尿素,20 mmol/L三羟甲基氨基甲烷(Tris)-盐酸缓冲液(pH 9.0)中,用Q-Sepharose FF和Sephacryl S-200HR树脂组成的色谱柱结合大部分溶解的膜蛋白质,然后采用氯化钠线性梯度洗脱蛋白质,分步收集后采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进一步分离洗脱组分的蛋白质。利用胶内胰蛋白酶消化技术将SDS-PAGE胶内分离的蛋白质降解为相应的肽段,然后以反相高效液相色谱分离和离子阱质谱仪鉴定肽段。根据文献报道和蛋白质的功能分类,在所鉴定的392个蛋白质中有306个可能为膜蛋白质或膜结合蛋白质。蛋白质的疏水性计算表明,GRAVY(grand average of hydropathicity)得分大于或等于0.00的蛋白质有83个。综上所述,我们有理由认为本实验方法基本符合小鼠肝脏膜蛋白质组学研究的要求。