Reduction of proteins during sample preparation and two-dimensional gel electrophoresis of woody plant samples

Protein extraction procedure and the reducing agent content (DTT, dithioerythritol, tributyl phosphine and tris (2-carboxyethyl) phosphine (TCEP)) of the sample and rehydration buffers were optimised for European beech leaves and roots and Norway spruce needles. Optimal extraction was achieved with 100 mM DTT for leaves and needles and a mixture of 2 mM TCEP and 50 mM DTT for roots. Performing IEF in buffers containing hydroxyethyldisulphide significantly enhanced the quality of separation for all proteins except for acidic root proteins, which were optimally focused in the same buffer as extracted.     

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea–urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.     

A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure

We report the results of a systematic investigation to quantify the losses of protein during a well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([(14)C]bovine serum albumin and a homogenate prepared from the liver of a rat that had been injected with [(35)S]methionine) were used, and recovery was quantified by digesting pieces of gel in H(2)O(2) and subjecting the digests to liquid scintillation counting. When samples were loaded onto the first dimension immobilised pH gradient strips by in-gel rehydration, recovery of protein from the strips was 44-80% of the amount of protein loaded, depending on the amount of protein in the sample. Most of the unrecovered protein appeared to have adhered to the reswelling tray. Losses during isoelectric focusing (IEF) were much smaller (7-14%), although approximately 2% of the protein appeared to migrate from sample strips to adjacent blank strips in the focussing apparatus. A further 17-24% of the proteins were lost into the buffers during equilibration prior to running in the second dimension. Losses during the second dimension run and subsequent staining with SYPRO Ruby amounted to less than 10%. The overall loss during 2-DE was reduced by approximately 25% when proteins were loaded onto the IEF strips using sample cups instead of by in-gel rehydration. These extensive and variable losses during the 2-DE procedure mean that spot intensities on 2-DE gels cannot be used to derive reliable, quantitative information on the amounts of proteins present in the original sample.     

Evaluation of an effective sample prefractionation method for the proteome analysis of breast cancer tissue using narrow range two-dimensional gel electrophoresis

One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.     

Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis

Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants. Therefore, the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation. Efficient solubilization of proteins strictly depends on the chaotrope and detergent in the extraction buffer. Despite the large number of detergents that have been developed for the use in protein extraction and IEF, there is no single compound able to efficiently extract proteins from any source. Hence, optimization has to be performed for each type of sample. We tested several chaotrope/detergent combinations to achieve optimal solubilization and separation of proteins from Norway spruce [Picea abies (L.) H. Karst.] needles and European beech (Fagus sylvatica L.) leaves and roots. The same chaotrope mixture (7 M urea, 2 M thiourea) was found to be suitable for the extraction and separation of proteins from all samples. Nonetheless, the efficiency of the surfactants tested varied between samples so that optimal extraction and separation was achieved with different detergents or combination of detergents for each sample. The 2-DE separation of spruce needle proteins was optimal in a mixture of two zwitterionic detergents (2% CHAPS and 2% decyl dimethylammonio propanesulfonate). Beech proteins were best separated in buffers containing sugar-based detergents (2% n-octyl beta-D-glucopiranoside in the case of leaf samples and 2% dodecyl maltoside for the root samples). IEF was performed in buffers with the same composition as the extraction buffer except for the root proteins that were better focused in a buffer containing 2% CHAPS.     

适用于黄麻根部蛋白质组学分析的双向电泳技术

以黄麻品种'9511'幼苗为试验材料,研究其根部蛋白提取方法的得率及不同的蛋白样品溶解方法、电泳上样量和IPG胶条pH范围对双向电泳图谱的影响.结果表明:采用三氯乙酸(TCA)/丙酮沉淀法提取黄麻根部蛋白质,蛋白得率为80 mg/g;蛋白粉末溶解采用两次水化法,裂解液中含有7 mol/L尿素、2 mol/L硫脲、4% CHAPS、65 mmol/L DTT、0.2%载体两性电解质和1 mmol/L PMSF,能够较充分地溶解蛋白质,且制备的样品浓度能够满足双向电泳上样要求;上样量为400 μg时得到的图谱分辨率高、蛋白斑点分布均匀、清晰;等电聚焦(Isoelectrofocusing,IEF)采用pH 4～7、17 cm的IPG胶条时所得图谱质量最佳.研究表明,样品的制备及IEF有效除盐对获得理想的2-DE图谱非常关键;取材、染色等细节对2-DE的重复性影响很大.     

不同上样方式对蛋白质组双向电泳图谱质量的影响比较

蛋白质组技术难点之一是如何获取尽可能多的细胞或组织的蛋白信息.双向电泳蛋白斑点数目直接反映了实验蛋白质组信息的完整性.除样品制备外,蛋白上样方式对双向电泳图谱的质量和完整性有直接的影响.实验论文从以下3个方面考察不同的蛋白上样方式对双向电泳图谱的影响;即：水化上样与杯上样;一次上样与重复上样;以及酸性端加样与碱性端上样.实验结果发现;蛋白上样量较大时,杯上样方式的图谱斑点数目较水化上样方式明显增多;样品蛋白浓度较高时,稀释多次上样明显优于一次性浓缩蛋白上样;蛋白裂解液(MCF7乳腺癌细胞)在酸性端加样对偏碱性蛋白的分离未发现明显优势.相反,在等电聚焦伏小时(Vh) 足够的前提下,碱性端加样对偏碱性端蛋白反而有利,表现为斑点数目较多,而且等电点方向拖尾减轻.实验结果对提高双向电泳的质量以及相关蛋白质组信息的完整性提供了有益的技术参考.     

棉花纤维蛋白质3种提取及二维电泳方法的比较

高质量的蛋白样品制备是进行二维电泳的先决条件.棉花纤维中含有纤维素、多酚、多糖等严重干扰二维电泳的物质, 增加了蛋白提取和二维电泳的难度.分别采用3种提取植物组织蛋白的方法(水法、酚法和尿素法), 提取棉纤维总蛋白, 进而进行了二维电泳分析.在蛋白产量、蛋白纯度和电泳图谱等方面对3种方法进行了比较, 结果采用酚法提取的样品取得了较好的电泳图谱, 有望成为从棉纤维样品中提取总蛋白的可选方法.     

红色红曲菌蛋白质组双向凝胶电泳体系的建立

红曲菌是一种具有较高食用和药用价值的丝状真菌,能够产生红曲色素、莫纳可林K等多种生理活性物质。通过分析比较培养基、蛋白质裂解液组成以及水化上样条件对双向电泳结果的影响,建立了红色红曲菌蛋白质组的双向凝胶电泳体系,为从蛋白质水平研究红曲菌及其次级代谢产物的生物合成提供依据。结果表明：用YES培养基培养红色红曲菌6 d,TCA 丙酮法提取菌体总蛋白质,蛋白质裂解液组分为8mol/L尿素,2mol/L硫脲,4 % CHAPS,1 % DTT和2 % Bio-lyte,可获得蛋白质样点数量多,清晰度高的双向电泳图像,为进一步研究红曲菌蛋白质组奠定了基础。     

Optimised two-dimensional electrophoresis procedures for the protein characterisation of structural tissues

The protein analysis of structural tissues is typically highly problematic. Amniotic membrane displays unique wound healing and anti-scarring properties; however, little is known concerning its active protein content. The structural nature of amniotic membrane necessitated development and extensive optimisation of the entire two-dimensional (2-D) workflow. Proteins were extracted using powerful solubilisation buffers and analysis carried out using 2-D electrophoresis followed by mass spectrometry (MS) identification. Preservation and processing resulted in prefractionation of soluble from structural and membrane-associated proteins. Enhanced protein solubility was achieved by cysteine blocking using both N,N-dimethylacrylamide (DMA) alkylation and bis(2-hydroxyethyl) disulphide (HED); an alternative procedure for the effective application of HED is demonstrated. The benefits of precipitation and cup-loading versus in-gel rehydration were also assessed, with procedures for the employment of HED with the latter described. Following optimisation, a representative sample 21 proteins were identified from amniotic membrane using MS verify procedures were MS-compatible. Our results demonstrate that techniques for the reproducible separation of proteins from a proteinaceous structural tissue have been optimised. Briefly, proteins are extracted using a thiourea/urea extraction buffer containing carrier ampholytes, dithiothreitol (DTT), and 3-(cyclohexylamino)-1-propanesulfonic acid (CHAPS). After DMA alkylation, proteins were precipitated (using the 2-D clean-up kit from Amersham Biosciences) and resolubilised in extraction buffer containing a lower concentration of DTT. Samples were either cup-loaded onto rehydrated HED-containing strips or rebuffered into HED-containing buffer followed by in-gel rehydration.     

Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.     

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.     

适用于盐生植物的双向电泳样品制备方法

比较了三氯乙酸,丙酮沉淀法（TCA）、三氯乙酸沉淀法（E-TCA）和酚抽法（Phe）3种方法对盐生植物盐角草（Salicornia europaea L．）总蛋白的提取效果。3种方法分别得到579、343和535个蛋白点;TCA和E-TCA法所得图谱均存在严重的横向纹理,Phe法所得图谱则背景干净,基本上没有纹理。说明Phe法不仅能很好地提取盐角草蛋白,而且能有效去除样品中的盐分。对Phe法的提取液进行了改进,所得图谱背景更加清晰,蛋白点数增加。为其他盐生植物以及嗜盐微生物蛋白质的提取提供了重要参考。     

A quantitative and qualitative method for direct 2-DE analysis of murine cartilage

Direct 2-DE analysis of cartilage is difficult due to the high proteoglycan content. Proteoglycan removal before IEF may however cause the partial or total loss of specific proteins making this approach ineffective when quantitative data are required to investigate protein expression differences. Thus, we have developed a 2-DE method including passive rehydration loading that does not require sample pretreatment and allows direct protein expression studies in cartilage samples.     

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

Analysis of the Shewanella oneidensis proteome by two-dimensional gel electrophoresis under nondenaturing conditions

Proteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two-dimensional gel electrophoresis (2-DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2-DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2-DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR-1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.     

西花蓟马蛋白的提取及双向电泳体系的建立

【目的】蛋白样品的制备是获得良好双向凝胶电泳(2-DE)图谱的前提,建立合理的西花蓟马蛋白的双向电泳体系,获得分辨率较高、重复性较好的图谱,能够为后续的研究提供有力支撑。【方法】实验以西花蓟马成虫为实验材料,对比了饱和酚法、TCA/丙酮法和直接裂解法3种蛋白提取方法,从中选出最适宜双向电泳分析的一种蛋白提取方法。【结果】3种方法蛋白提取率差异显著,直接裂解法蛋白提取率最高,饱和酚法的蛋白提取率最低;3种方法的SDS-PAGE条带数差异不明显;TCA/丙酮法的双向凝胶图谱效果最好,蛋白点最多。【结论】TCA/丙酮法能够有效去除西花蓟马蛋白中的干扰物质,是最适合西花蓟马双向凝胶电泳的蛋白提取方法,为后续西花蓟马在蛋白组学方面的研究奠定了基础。     

Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments

To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.     

口虾蛄性腺组织蛋白质双向电泳体系的建立及优化

旨在通过口虾蛄雄性、雌性性腺组织蛋白质双向电泳技术体系的优化,获得雄性、雌性口虾蛄性腺蛋白质的表达图谱。结果表明,不同的蛋白提取方法,上样前处理方法,上样量,聚焦时间及雌、雄性口虾蛄性腺蛋白表达图谱存在一定的差异。雌性、雄性口虾蛄用Tris-HCl提取后用丙酮沉淀方法提取蛋白质、不经上样缓冲液处理、上样量为10μg时,得到较好图谱。对图谱分析发现在pH4-6.5范围内,雌性口虾蛄性腺可溶性蛋白质种类多于雄性。雄性口虾蛄蛋白质点相对于雌性较少,且蛋白质大部分分布于酸性端。经过蛋白质双向电泳体系的优化,能显著提高双向电泳图谱的分辨率,为进一步研究口虾蛄性别差异表达蛋白的筛选,后续的口虾蛄蛋白质组学研究提供技术保障。