一种提高固定pH梯度（IPG）凝胶双向电泳的重复性、分辨率和通量的方法

在蛋白质组学研究中 ,双向聚丙烯酰胺凝胶电泳是现行蛋白质分离的最重要的方法之一。实验发展了一种提高固定pH梯度 (IPG)凝胶双向电泳的重复性、分辨率和通量的方法 :在一块SDS 聚丙烯酰胺凝胶上同时进行多块固定pH梯度(IPG)凝胶 (Multi stripsononeSDSgel,MSOG)电泳。用此方法比较了人肝癌细胞、不同生长状态的人肝癌细胞、3T3细胞的蛋白质以及同一个样品在不同大小的第二向凝胶系统 (大型和中型凝胶 )的双向电泳图谱。结果表明 ,同一样品在 13cmIPGStrip双向电泳可分离 2 0 0 0以上蛋白质点且图谱蛋白质点的匹配率可超过 95 %以上。同时又可以最大程度地降低凝胶背景对蛋白质点比较分析的干扰 ,从而提高了双向电泳分离蛋白质的分辨率和通量。这些优点都有助于差异蛋白质组学特别是细胞器差异蛋白质组学研究的自动化。     

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.     

Sample prefractionation with Sephadex isoelectric focusing prior to narrow pH range two-dimensional gels

Due to their heterogeneity and huge differences in abundance, the detection and identification of all proteins expressed in eukaryotic cells and tissues is a major challenge in proteome analysis. Currently the most promising approaches are sample prefractionation procedures prior to narrow pH range two-dimensional gel electrophoresis (IPG-Dalt) to reduce the complexity of the sample and to enrich for low abundance proteins. We recently developed a simple, cheap and rapid sample prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated gels. Complex sample mixtures are prefractionated in Sephadex gels containing urea, zwitterionic detergents, dithiothreitol and carrier ampholytes. After IEF, up to ten gel fractions alongside the pH gradient are removed with a spatula and directly applied onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of two-dimensional (2-D) gel electrophoresis. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and the full compatibility with subsequent IPG-IEF, since the prefactionated samples are not eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with subsequent IPG-IEF. Prefractionation allows loading of higher protein amounts within the separation range applied to 2-D gels and facilitates the detection of less abundant proteins. Also, this system is highly flexibile, since it allows small scale and large scale runs, and separation of different samples at the same time. In the current study, this technology has been successfully applied for prefractionation of mouse liver proteins prior to narrow pH range IPG-Dalt.     

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.     

人肺巨细胞癌蛋白质组的二维电泳和计算机图象分析

为优化用于蛋白质组研究的二维电泳技术和计算机图象分析技术 ,以及初步分析比较与肿瘤细胞转移相关的蛋白质 ,以人肺巨细胞癌 (PLA- 80 1 - D、C)高、低转移株作为研究对象 ,应用 IPG-phor进行第一向等电聚焦 ,随后 ,在 Protein IPG conversion Kit上进行垂直 SDS- PAGE的分离 .利用光密度仪对银染的凝胶扫描 ,通过 PDQuest软件进行蛋白斑点检测和配比 .结果表明 :(1 )应用 IPGphor,采用样品直接加入重泡胀溶液的形式 ,增大了溶解性 ,缩短聚焦时间、增大样品负荷量 (分析型 ) ,提高了分辨率 .(2 )比较宽 (p H=3～ 1 0 L)、窄 (p H=4～ 7L)范围 IPG胶条 ,窄 p H范围的 IPG胶条具有较高的分辨率 .(3)比较 PLA- 80 1 - C、D细胞蛋白图谱之间的差异 ,其相关系数为 0 .7339± 0 .0 2 91 ;仅在 PLA- 80 1 - C株出现的蛋白为 1 79个 .     

用于双向凝胶电泳分析的奶牛乳清蛋白样品制备方法的比较

为建立适用于双向凝胶电泳分析的奶牛乳清蛋白的制备方法,分别比较了直接裂解法、三氯乙酸-丙酮法,Trizol法和2-D clean up kit法对奶牛乳清蛋白提取效率和双向凝胶电泳图谱的影响.用2-D Quant Kit试剂盒测定蛋白浓度,分别用十二烷基磺酸钠 聚丙烯酰胺凝胶电泳和双向凝胶电泳进行奶牛乳清蛋白的分离.蛋白定量结果表明,2-D clean up kit法产率最高,直接裂解法、三氯乙酸-丙酮法次之,trizol法产率最低;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳结果表明,2- D clean up kit法提取的蛋白质量最高;双向电泳图谱分析表明,2-D clean up kit法得到的蛋白图谱与另外3种方法相比,检测到的蛋白点最多,图谱背景清晰,分辨率最高.结果提示,2-D clean-up法相对最适合于双向凝胶电泳分析奶牛乳清蛋白样品的制备,尤其对一些低丰度高分子量蛋白的分离效果较为明显.     

Comparative 2-DE protein analysis in a 3-D geometry gel

Two-dimensional gel electrophoresis (2-DE) separation has not been considered suitable for large-scale comparative protein expression studies due to its limited throughput. We present a high-throughput analysis method based on three-dimensional (3-D) geometry gel electrophoresis. Following conventional isoelectric focusing (IEF), up to 36 immobilized pH gradient (IPG) strips are arrayed on the top surface of a 3-D gel body, and the samples transferred electrokinetically to the gel. A specific thermal management ensures that sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) occurs under identical electrophoretic and thermal conditions, avoiding gel-to-gel variations and thereby providing immediate comparability of the separation patterns. Proteins are Cy3-labeled for online detection of laser-induced fluorescence (LIF). Images are acquired by a digital camera and recorded as a 3-D image stack during electrophoresis. Image processing software decomposes the 3-D image stack into vertical sections representing conventional 2-DE slab gels, making results immediately accessible without further gel processing. The large number of simultaneously analyzed samples (n = 36) allows treating the sample index as a quasi-continuous experimental parameter (e.g., concentration, time, dose). The method offers a wide range of applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology, like protein monitoring during disease development and screening of drug candidates for their effect on protein expression.     

Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager

Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both after isoelectric focusing (IEF) and PAGE with a sensitivity higher than or comparable to standard silver staining methods. In addition to the post-labeling methods mentioned above, pre-labeling with the cysteine-specific fluorophore monobromobimane before 2-D PAGE is a sensitive, fast, and cost-effective alternative to existing staining protocols.     

Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips

By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.     

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。     

Isoelectric point-based prefractionation of proteins from crude biological samples prior to two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 min using an anion-exchange resin and two expressly designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pI values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially expressed proteins were greatly improved.     

G-electrode-loading method for isoelectric focusing, enabling separation of low-abundance and high-molecular-mass proteins

The G-electrode-loading method (GELM) is a technique enabling a large number of proteins from rat liver to enter an immobilized pH gradient (IPG) gel strip for isoelectric focusing (IEF). In this method, three slips containing the sample solution are placed on the cathodic edge of an IPG gel strip and a slip containing Chaps solution, a filtration membrane, and an electrode slip are placed on top. Finally, a G-electrode is placed on these slips. The Chaps solution (an amphoteric compound) is supplied gently to the sample solution during IEF and helps the proteins in the sample solution to enter the IPG gel strips with a high solubilization capacity. This method was compared with traditional slip-loading and in-gel rehydration, and it showed the best results for protein separation, including high-molecular-mass proteins.     

Optimization of IPG strip equilibration for the basic membrane protein mABC1

Many proteins with extreme physical properties, including basic and acidic proteins, membrane proteins, and very large proteins, present specific challenges to 2-DE separation. Using a pressure-blotting approach, we demonstrate that a basic integral membrane protein, mitochondrial ATP-binding cassette protein 1 (mABC1), focuses in the IPG strip, but fails to enter into the 2-D SDS-PAGE gel. Through modifying the equilibration conditions between the IPG strip and 2nd dimension, we demonstrate that only by increasing both the volume (from 3 to 6 mL for a 7-cm strip) and SDS concentration (from 2 to 10%) of the equilibration buffer is migration of mABC1 into the 2nd dimension achieved. While 2-DE remains one of the core separation technologies of proteomic analysis, proteins that are extremely basic, hydrophobic, or of large mass present significant challenges to 2-DE separation due to aggregation, oxidation, precipitation, and the physical limitations of the 1-D IPG strip. Since the advent of commercially available IPG strips, numerous groups have experimented with IEF conditions using various detergents alone or in combination, alternative denaturants, and thiol oxidation agents to improve protein focusing. Effective 2-DE separation of membrane proteins has been affected dramatically by these advances in protein solubilization, as well as improvements in isolation of membranes, delipidation, and active in-gel rehydration. Since the development of commercially available basic IPG strips, the most significant advance in the separation of basic proteins has been the introduction of hydroxyethyldisulfides, either alone or in combination with DTT. While hydrophobic proteins were once virtually absent from the 2-D gel, and basic proteins were only visible as dense streaks, now many groups are undertaking large-scale analyses of membranes and basic proteins. Using this base of experimental tools, we embarked on a proteomic analysis of cardiac mitochondrial membranes, hoping to combine the knowledge gained from ongoing targeted protein chemistry and molecular biology studies with a broader-based proteomic analysis. Of particular interest is the inner mitochondrial membrane protein mABC1 (mitochondrial ATP-binding cassette protein 1), which may play a significant role in cardioprotection as part of the mitochondrial ATP-sensitive potassium channels. Therefore, in designing our 2-DE approach, it was crucial to ensure that mABC1 is focused, observable, and quantifiable, despite being an integral membrane protein of pI 9.37.     

nm23-H1基因转染前后人高转移大细胞肺癌细胞株双向凝胶电泳图谱分析

为了更全面地了解nm23-H1在肺癌中发挥转移抑制的机理,用双向凝胶电泳技术比较人高转移大细胞肺癌细胞株(L9981)和转染nm23-H1基因的人大细胞肺癌细胞株(L9981-nm23-H1)间蛋白表达的差异.利用固相pH梯度双向凝胶电泳分离人高转移大细胞肺癌细胞株(L9981)和转染nm23-H1基因的人大细胞肺癌细胞株(L9981-nm23-H1)的总蛋白,用图像分析软件比较分析以识别细胞间的差异表达蛋白质.结果成功地获得了两株细胞蛋白组分辨率高、重复性好的双向凝胶电泳图谱.软件分析两种细胞的凝胶电泳图谱后发现,在相同分析条件下识别的蛋白质斑点数L9981为902±169个、L9981-nm23-H1为1160±212个.比较L9981和L9981-nm23-H1人大细胞肺癌细胞株的双向凝胶电泳蛋白质图谱后发现6个蛋白质点仅在L9981中有表达,17个蛋白质点仅在L9981-nm23-H1中有表达.此外,发现13个在两种细胞株中均存在,但表达量差异在2倍以上的蛋白质点(P<0.05).结果提示,nm23-H1基因转染引起人高转移大细胞肺癌细胞株蛋白质表达谱的变化,可能是其逆转肺癌侵袭转移的生物学基础.     

A universal method for two-dimensional polyacrylamide gel electrophoresis of membrane proteins using isoelectric focusing on immobilized pH gradients in the first dimension.

Two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension, initially applied for the separation of soluble and total cellular proteins, has been extended to the analysis of membrane proteins. We show that the usual procedures lead to artifacts and irreproducible results due to aggregation and precipitation of proteins and protein-phospholipid complexes during isoelectric focusing (first dimension) and sodium dodecyl sulfate (SDS) gel electrophoresis (second dimension). Optimized solubilization procedures for hydrophobic membrane proteins are presented and the use of dilute samples is shown to be essential to overcome the major problems in isoelectric focusing. Increased volumes of samples dissolved in rehydration buffer are applied by direct rehydration of dry immobilized pH gradient (IPG) gels. Isoelectric focusing in 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) without urea gives good results as does 2% Nonidet-P40 with 8 M urea. Heat denaturation should be avoided. An optimized equilibration procedure for IPG gel strips in SDS sample buffer prior to separation in the second dimension was developed that minimizes loss of proteins and results in high-resolution two-dimensional electropherographic maps with a minimum of streaking. The gel strips are partially dehydrated at 40 degrees C and shortly reswollen in situ on the SDS slab gel in SDS-sample buffer containing agarose.     

Immunodepletion of albumin for two-dimensional gel detection of new mouse acute-phase protein and other plasma proteins

Immunodepletion of albumin to improve the 2-D gel resolution of human plasma proteins has recently been described. With the importance of mouse models in many studies in which serum or plasma is often analyzed, we have adopted this approach to immunoprecipitate mouse albumin and evaluated its effectiveness for 2-D separation of mouse plasma proteins. Purified polyclonal antibodies against mouse albumin were effective depleting intact albumin as well as its numerous fragments from mouse plasma samples. Removal of albumin resulted in better resolution of mouse plasma proteins. Three proteins, alpha2-macroglobulin, coagulation factor XII, and hemopexin, that were previously either undetectable or poorly resolved, were identified from albumin-depleted 2-D gels by peptide mass fingerprinting. Albumin depletion also led to partial loss of several other proteins such as clusterin and gelsolin. This loss can be attributed to the interaction with albumin itself because the specificity of the antibody was demonstrated by Western blot. When applying this method to the 2-D separation of plasma from inflamed mouse induced by cutaneous burn injury with superimposed Pseudomonas aeruginosa infection, the upregulation of inter alpha-trypsin inhibitor heavy chain 4 (ITIH4) and hemopexin was unambiguously detected along with other mouse acute-phase proteins (APP), including haptoglobin and serum amyloid A. Based on the significant increase of ITIH4, we propose that this protein is a new member of mouse APP that are upregulated during the inflammatory response.     

Problems encountered in measuring erythrocyte pyrimidine 5'-nucleotidase activity

An improved method of two-dimensional electrophoresis that allows unequilibrated first-dimension gels to be loaded and electrophoresed on second-dimension gels twice the length used in the O'Farrell technique has been developed. Normally, the electrophoresis of unequilibrated first-dimension gels on long second-dimension gels with the resolving gel set at pH 8.8 results in poor resolution of low-molecular-weight proteins. Adjusting the pH of the resolving gel to pH 8.3 maintains the low-molecular-weight proteins in a stacked configuration during their migration through the length of the 10% acrylamide gel. Utilization of a 10 to 20% exponential polyacrylamide gradient in a resolving gel set at pH 8.3 separates these low-molecular-weight proteins with excellent resolution. Electrophoretic resolution of protein spots in resolving gels set at pH 8.8 is not as sharp as in gels set at pH 8.3, and resolution progressively deteriorates in gels set at higher pH values.     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

Development of narrow immobilized pH gradients covering one pH unit for human seminal plasma proteomic analysis

Micropreparatively loaded two-dimensional (2-D) electrophoresis gels were used to identify human seminal plasma polypeptides by using narrow immobilized pH gradients (IPGs) covering one pH unit as first dimension. This investigation was restricted to IPG 4.5-5.5 and 5-6 because of the main spot distribution in the acidic part of the 2-D map performed with IPG 3-10, a zone presumed to be rich in spermatogenic markers. Both highly expressed and minor spots of the 2-D map were analyzed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectroscopy analysis. Identification was obtained by a combination of mass spectrometry and database searching. Identified proteins were of different origin from the male genital tract and some proteolysis was observed. They appeared as either isolated molecules or isoforms. At the analytical level, narrow IPGs allowed a two-fold increase in the number of spots, improved resolution of minor spots particularly in surrounding of highly expressed spots and sensitivity level. Some of these faint spots were differently expressed in azoospermic patients as compared to normospermic men. Therefore, zooming-in on the proteome of human seminal plasma allowed more accurate differential expression analysis of impaired spermatogenesis associated markers.