水牛精子蛋白质组双向电泳体系的建立和优化

建立和优化一种适合水牛精子蛋白质组学研究的双向电泳技术。以水牛精子为研究对象,比较两种不同配方的裂解液,以及不同上样量对其2-DE图谱质量的影响。结果显示,以7 mol/L尿素、2 mol/L硫脲、4%CHAPS、1%DTT、0.5%Cocktail of protease inhibitors为裂解液,24 cm胶条上样量200μg时,可获得较好的精子总蛋白质2-DE图谱。运用ImageMaster 2-Dplatinum分析软件检测出约500个蛋白质点,蛋白质大部分分布在等电点5-7之间,分子量范围约40-90 kD。     

牛精子蛋白质组学技术平台的建立及冻融前后精子差异蛋白初步分析

本研究通过探索不同的精子蛋白制备方法、水化液成分和优化2D电泳程序以建立牛精子蛋白质组学研究技术平台,同时以牛鲜冻精为实验材料通过差异凝胶电泳寻找冻融前后精子蛋白的改变。结果表明：使用改进的热TRIzol法裂解精子细胞制备蛋白,结合优化的2D电泳技术可建立稳定的牛精子蛋白质组学研究技术平台。差异凝胶电泳揭示牛精子在冻融后有质和量的改变：冻融后缺失的蛋白点有20个,表达下调的有2个,表达上调的有10个。作为一项阶段性的实验成果,本研究建立的2D平台和所发现的冻融引起的差异表达蛋白质点为揭示冷冻损伤机理和性控精液的差异蛋白质组学研究奠定了较好的基础。     

hLCN6单克隆抗体偶联免疫磁珠用于混合细胞中精子的分离与法医学鉴定

hLCN6 (Human lipocalin 6)是附睾特异性分泌蛋白,它能与精子结合,对精子的成熟发挥着重要作用。为了探究应用抗hLCN6单克隆抗体偶联免疫磁珠技术分离混合细胞中精子的可行性,建立混合斑中精子细胞分离的新方法,制备了不同比例的精子-上皮细胞混合悬液及混合斑样本,以生物素标记的hLCN6单克隆抗体孵育样品,再用亲和素包被的免疫磁珠捕获分离精子细胞,提取精子DNA进行PCR-STR (Short tandem repeat)分型,同时以差异裂解法提取精子,比较两者的差异。经ELISA检测,hLCN6单克隆抗体与抗原的亲和力解离常数(Kd)为3.47×10~–9 mol/L。免疫印迹和免疫荧光结果显示,hLCN6在精子中可检测到,主要定位于精子头部的顶体后区域,但不能在上皮细胞中检测到。hLCN6抗体偶联的免疫磁珠复合物能够实现精子细胞的捕获和分离,显微镜观察显示免疫磁珠能够与精子头部特异性结合。对精子个数为10~3/mL的混合悬液,STR分型成功率(正确分型13个以上,RFU200)为90%。当精子数量≥10~4/mL时,分型成功率达10~0%;对精子数分别为10~3/mL、10~4/mL、10~5/mL的混合斑STR分型成功率分别为40%、90%和10~0%。综上,hLCN6抗体偶联免疫磁珠法可以有效分离混合细胞中的精子,分型成功率高于传统的差异裂解法。该方法简单高效,可作为性侵案件中法医混合斑检验的有效补充手段。     

适合水稻悬浮培养细胞蛋白质组分析的双向电泳技术

水稻是研究植物蛋白质组学的一个重要试材,从蛋白质的提取,裂解缓冲液的成份和浓度,第一向聚焦的条件,蛋白上样量和二向胶的选择方面,对水稻悬浮培养细胞的双向电泳条件进行了比较和优化。结果表明,采用甲醇/氯仿沉淀蛋白,裂解缓冲液中加入0.5%的两性电解质、并加入盐桥,17cm的胶条、850μg的上样量可达到较好的等电聚焦效果;8%-16%梯度胶分离蛋白较为适宜。     

黄瓜悬浮细胞蛋白质组双向电泳分析技术

为建立适于黄瓜悬浮细胞蛋白质组分析的双向电泳体系,对黄瓜悬浮细胞蛋白质双向电泳分析所采用的胶条pH范围、样品制备方法、裂解液配方及分离胶浓度等参数进行研究。结果表明,采用pH范围为4～7的IPG胶条,直接裂解后丙酮沉淀法制备黄瓜悬浮细胞蛋白质,裂解液为8mol/L尿素、2mol/L硫脲、2%IPG Buffer、4%CHAPS、1%TBP、65mmol/L DTT、2mmol/L EDTA、0.001%溴酚蓝和1%鸡尾酒,分离胶浓度为11%,可获得蛋白质点分离清晰的双向电泳图谱。     

缺铁逆境胁迫下水稻叶片蛋白的双向电泳及其蛋白质组图谱分析

水稻幼苗经缺铁胁迫诱导分别处理1、3、5天后,用酚法和TCA/丙酮法提取叶片中的可溶性蛋白进行双向电泳分析,从而研究在缺铁条件下叶片中蛋白表达的动态变化规律.结果显示1.不同pH IPG胶条分离蛋白的效果不同.用pH3-10的IPG胶条进行双向电泳,经考马斯亮蓝染色后,可在胶面上检测到大约450个蛋白点,其中约有89%的蛋白是酸性蛋白.如果用pH4-7的IPG胶条进行双向电泳,则可检测到大约600个蛋白点,其中有29个蛋白是上调表达,1个蛋白是下调表达,5个蛋白是诱导特异表达.2.不同方法提取的可溶性蛋白质量不同.TCA法简单易操作,似乎对于碱性蛋白的抽提效果更好,在2-DE图像上,减性端显示的蛋白点多;但此方法所得蛋白的再溶性差.酚法提取的蛋白再溶性好,所抽提的蛋白量较大,纯度较高.     

沙漠小球藻的蛋白双向电泳分析

为建立适用于小球藻(Chlorellasp.TLD6B)蛋白质组分析的双向电泳体系,该研究比较了TCA/丙酮沉淀法和Trisol提取法对小球藻蛋白的提取效果,不同pH梯度IPG胶条(pH3~10和pH4~7)、不同蛋白质上样量、不同聚焦程序对小球藻蛋白的分离效果。结果表明:(1)采用Trisol提取法可获得较高纯度蛋白,当选择24cm pH 3~10的线性IPG胶条时,上样量为500μg,聚焦80 000Vh效果最佳,可分辨蛋白点达726个;当选择24cm pH 4~7的线性IPG胶条时,上样量为1 000μg,聚焦80 000Vh效果最佳,可分辨蛋白点达1 230个。(2)该实验随机挑选了10个胶内蛋白点进行MALDI-TOF/TOF-MS鉴定分析表明,其中8个蛋白点鉴定成功,进一步说明Trisol提取法可适用于小球藻双向电泳分析。     

一种适用于双向电泳分析的高效真菌线粒体提取及蛋白质制样方法

用液氮冷冻研磨法破碎真菌菌丝细胞,通过差速和蔗糖密度梯度离心分离纯化板栗疫病菌线粒体,所得线粒体产率(质量比)约为1/10~4。电子显微镜观察和蛋白印迹表明,所制备的线粒体完整性好,没有检测到其它细胞成分的污染。使用膜蛋白裂解液溶解线粒体制备蛋白样品,将蛋白样品200μg上样于pH3~10,24cm的非线性胶条进行等电聚焦,电聚焦后再进行第二向SDS-PAGE电泳分离,经银染获得重复性好、背景清晰、分辨率高(680±15个蛋白质点)的凝胶图谱。随机选择10个蛋白质点进行质谱分析,9个获得有效注释,均为线粒体特异性蛋白质,表明所制备的蛋白样品非常适合双向电泳分析及其后续的质谱鉴定。     

水牛睾丸曲精细管蛋白质组双向电泳方法的建立及优化

建立并优化了沼泽型水牛睾丸曲精细管蛋白质分离的双向电泳体系,为后续水牛睾丸曲精细管蛋白质表达谱的鉴定和研究奠定基础。比较不同IPG胶条(线性与非线性)、胶条pH范围和蛋白质上样量这三个参数对双向电泳结果的影响。结果显示,采用上样量350μg的曲精细管总蛋白、24 cm且pH值4～7的线性IPG胶条能够更好地分离水牛睾丸曲精细管蛋白质,获得质量较好且分辨率较高的双向电泳图谱,得到大约486个蛋白点。双向凝胶电泳技术能够对水牛睾丸曲精细管蛋白质进行有效分离,并通过对双向电泳体系的优化可获得蛋白质点清晰且分辨率更高的双向电泳图谱。     

小麦小花高纯度线粒体的分离及其蛋白质双向电泳体系建立

应用差速离心和Percoll不连续密度梯度法分离纯化小麦三核期小花线粒体. 在裂解液选择、IPG胶条pH值范围、SDS-PAGE胶浓度及蛋白质上样量等方面对线粒体蛋白质双向电泳体系进行探索和优化,确立了一套适用于小麦小花高纯度完整线粒体的分离方法及其蛋白质双向电泳的技术体系. 结果表明,采用20%、24%和40% Percoll密度梯度和28% Percoll自形成密度高速离心体系,获得了有活性、高纯度且较完整的线粒体;经TCA-丙酮法提取蛋白,以7 mol/L尿素,2 mol/L硫脲,4% CHAPS(W/V),65 mmol/L DTT,0.5% IPG缓冲液(V/V),0.001% 溴酚蓝(W/V)裂解液溶解蛋白,采用17 cm,pH 4～7 IPG胶条和11% SDS-PAGE分离胶,上样量为160 μg,硝酸银染色法,更适合小麦小花线粒体蛋白质组双向电泳分离. 经PDQuest 2DE 8.0.1软件包统计分析,在2-DE图谱上分辨出约150个蛋白点,蛋白点清晰呈圆形,无横条纹干扰,这为利用双向电泳技术在亚细胞水平对线粒体进行蛋白质组学研究与分析奠定了基础,更为进一步分析研究线粒体与雄性不育的关系提供了理论与技术支撑.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.     

High-sensitivity analysis of human plasma proteome by immobilized isoelectric focusing fractionation coupled to mass spectrometry identification

Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the analysis of proteins. Here, we described a strategy combining isoelectric focusing in immobilized pH gradient strips, and mass spectrometry to create a new high-throughput and sensitive detection method. Protein mixture is separated by in-gel IEF, then the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the resulted peptides are subjected to reversed-phase high performance liquid chromatography followed by electrospray-linear ion-trap tandem mass analysis. Using this optimized strategy, we have identified 744 distinct human proteins from an IPG strip loaded only 300 microg of plasma proteins. When compared with other works in published literatures, this study offered a more convenient and sensitive method from gel to mass spectrometry for the separation and identification proteins of complex biological samples.     

茶树叶片蛋白双向电泳体系建立与应用

为开展茶树Camellia sinensis 低温和干旱胁迫下差异蛋白的分离和鉴定,以抗逆性较强的茶树品种‘迎霜’为试材,通过对提取方法、IPG 胶条pH 范围、上样量、分离胶浓度、染色方法的比较,筛选适用于茶树叶片的蛋白质双向电泳体系。结果表明,采用TCA-丙酮法或Tris-HCl 法提取叶片总蛋白,选用17 cm pH 4~7IPG 胶条用于等电聚焦,选择1.6~2.2 mg 上样量、13.5%聚丙烯酰胺凝胶进行分离,随后通过高敏考马斯亮蓝R-250 法染色;最终,叶片各分子量的蛋白充分分离,获得的双向电泳图谱分辨率高、背景清晰、重复性好,适用于‘迎霜’低温和干旱胁迫下叶片差异蛋白分析。     

New allelic product of the PRH1 locus coding for salivary acidic proline-rich proteins

A new polymorphic acidic proline-rich protein (As) was found in human parotid saliva by SDS and basic polyacrylamide gel electrophoresis. The phenotypic relationships and family studies support the hypothesis that the As protein is another allelic product of the PRH1 locus. The As protein could not be discriminated from the parotid isoelectric focusing (PIf) protein by isoelectric focusing gel electrophoresis due to similar migration of the two proteins. In order to determine salivary PRH1 phenotypes it is necessary to use SDS or basic gel electrophoresis in addition to the isoelectric focusing gel electrophoresis. The As protein was not found in Caucasians. The allele frequencies of the PRH1 locus in Japanese were PRH1 (double-band protein) = 0.035, PRH1(2) (acidic protein) = 0.193, PRH1(4) (PIf) = 0.751, and PRH1(5) (As) = 0.021.     

Identity of SDS-insoluble Proteins with Purified Glutenin from Wheat Flour

Sodium dodecyl sulfate (SDS)-insoluble proteins from wheat flour were solubilized by the reduction of their disulfide linkages with 2-meracaptoethanol. The polypeptide compositions of the reduced SDS-insoluble proteins were compared with those of the reduced glutenin by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and amino acid analysis. SDS-polyacrylamide gel electrophoretic patterns of the reduced SDS-insoluble proteins almost coincided with those of the reduced glutenin. Seven major bands (Band 1–7) were obtained from both samples of the reduced proteins. These protein bands were subjected to analysis of amino acid compositions and isoelectric focusing, and similarities between polypeptides of the SDS-insoluble proteins and the glutenin were observed in their amino acid compositions and isoelectric focusing patterns. The results obtained suggested that the preparation of the reduced SDS-insoluble proteins might be used as a simple and rapid method to obtain the glutenin subunits.     

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.     

Purification of bicarbonate-sensitive sperm adenylylcyclase by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-affinity chromatography.

Anion transport inhibitors, such as SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) and heparin, inhibit reversibly the bicarbonate-sensitive adenylylcyclase of porcine sperm plasma membrane. In the light of this, SITS- and heparin-affinity chromatographies were applied in order to purify sperm adenylylcyclase. SITS-Affi-Gel 102 binds proteins extracted from the porcine cauda epididymal sperm plasma membrane by Lubrol-PX, more selectively than heparin-agarose. However, recovery of adenylylcyclase activity is higher when heparin-agarose is used. The hormone-sensitive liver adenylylcyclase, which is less sensitive to bicarbonate than sperm enzyme, has less affinity for these affinity resins than sperm enzyme. Adenylylcyclase can be purified to apparent homogeneity on two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) from the Lubrol-PX extract of the purified sperm plasma membrane by using SITS-affinity chromatography at the first step of the purification followed by preparative isoelectric focusing and gel filtration. The molecular weight and pI of the purified enzyme are 46,300 and 6.9, respectively. The purified enzyme activity is highly dependent on Mn2+. Bicarbonate activates even the purified enzyme both by decreasing Km and by increasing Vmax.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.