拟南芥蛋白质组研究中双向电泳技术条件的优化

双向电泳技术是蛋白质组学研究中的关键技术,是目前分辨率最高的工具之一.而提高双向电泳图蛋白质点的数目和分辨率,可以提高蛋白质组技术平台的信息完整性.通过对拟南芥双向电泳技术过程中的适当改进,如蛋白质的提取与溶解方法、上样量和聚丙烯酰胺凝胶浓度,加入硫脲,硫代硫酸钠等,对拟南芥双向电泳技术进行了优化,提高了双向电泳图谱的蛋白质点数目与分辨率.     

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.     

不同提取方法及上样量对牡丹试管苗茎基部蛋白双向电泳的影响

为建立一套适合于牡丹试管苗茎基部蛋白的双向电泳技术,以便更好地利用蛋白质组技术研究牡丹试管苗不定根的发生机理,本研究比较了三种不同蛋白质提取方法对双向电泳结果的影响,并在蛋白质上样量方面进行了比较。结果表明,乙酸铵/甲醇酚提取法所得2-DE图谱的蛋白点很少,仅检测到45个,且较模糊,有明显的拖尾现象,分辨率很低;乙醇/乙醚丙酮法所得的蛋白点也较少(101个),较模糊,且横竖纹干扰较大;三氯乙酸/丙酮法所得蛋白点数较多,可检测到434个清晰的蛋白点,且形状规则,重复性好,适合后续分析,操作也较为简便。用三氯乙酸/丙酮法提取蛋白,采用800μg、1000μg和1200μg三个不同的上样量进行双向电泳,在上样量为1200μg时(IPGpH3～10,24cm),蛋白质在12%SDS-PAGE胶上得到了较好的分离,在2-DE图谱上可分辨出562个蛋白点。因此,三氯乙酸/丙酮法是较适合于牡丹试管苗茎基部蛋白质提取的方法,1200μg是较为合适的上样量。     

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。     

大肠癌双向电泳分离条件的建立及其初步分析

采用固相pH梯度等电聚焦为第一向,SDS-PAGE垂直电泳为第二向建立了大肠癌双向电泳分离技术实验条件,对样品的处理、水化、等电聚焦、凝胶平衡等步骤进行了优化,成功地获得了大肠癌分辨率高、重复性好的双向电泳图谱.癌组织样品经3次重复实验共获得蛋白质斑点数1 186±46个,蛋白质斑点位置在IEF方向平均偏差为1.67±0.29 mm,在SDS-PAGE方向为1.41±0.16 mm,蛋白质表达量的相对标准差为6.67 %±2.25 %.经ImageMaster 2D Elite软件初步分析后发现了一些差异表达的蛋白质.     

油菜叶片总蛋白质双向电泳样品制备方法的改进

以甘蓝型油菜"扬油6号"的叶片为试验材料,分别采用传统的TCA/Acetone(三氯乙酸/丙酮沉淀法)和改进的PEG(polyethylene glycol)分步提取法提取叶片可溶性总蛋白,并利用条件一致的蛋白质双向电泳体系进行比较。TCA/Acetone法提取的蛋白质双向电泳图谱背景中由于高丰度"housekeeping"结构蛋白的存在,特别是叶片中参与光合作用的Rubisco蛋白的干扰,图谱中低丰度调控蛋白受到了高度覆盖和遮蔽现象,影响双向电泳图谱的质量。而PEG分步提取法提取的蛋白质样品,可以剔除Rubisco蛋白,使获得的双向电泳图谱清晰,无斑点间的遮蔽现象,为油菜叶片蛋白质组定量和定性分析提供了丰富的信息。     

甜瓜叶片中Rubisco的去除及双向电泳体系的优化

为降低Rubisco的干扰,建立适合于甜瓜叶片蛋白质的双向电泳技术,本文比较了不同全蛋白提取方法和上样量对双向电泳的影响。结果表明,Mg/NP-40/PEG3350/TCA/丙酮提取法可去除样品中绝大多数的Rubisco,使低丰度蛋白得以检测,适合后续分析。以该法提取的蛋白,采用600、800、1000和l200μg四种上样量进行双向电泳,上样量为l000μg的样品用pH3～10的IPG胶条,在2-DE胶上分辨出质量好、数量多的蛋白质点（562个）。因此,Mg/NP-40/PEG3350/TCA/丙酮法是适合甜瓜叶片蛋白质提取的方法,l000μg是最适上样量。     

蝴蝶兰叶片蛋白质提取及双向电泳体系优化

通过对蛋白质提取、IPG胶条选择、上样量、水化方式、聚焦条件等方面的优化,建立蝴蝶兰叶片蛋白质的双向电泳体系。结果表明,采用酚抽提法提取蝴蝶兰叶片蛋白质的纯度较高,复溶较完全;双向电泳优化体系选用24 cm pH 3～10 NL的IPG胶条,被动水化,上样量为1.35 mg,B1程序进行等电聚焦,12%分离胶进行第二向电泳,考马斯亮蓝G-250染色。该方法获得分辨率较高、重复性较好的蝴蝶兰叶片双向电泳图谱,蛋白数点多达1163个,可以满足蝴蝶兰蛋白质组学研究和分析。     

芒果叶片双向电泳体系的建立及优化

为建立适用于芒果叶片总蛋白的双向电泳(two-dimensional electrophoresis,2-DE)体系,使用3种方法(尿素/硫脲法,酚法和三氯乙酸/丙酮法)提取芒果叶片蛋白,采用2种水化方式(主动水化和被动水化)和4种样品上样量(1 000μg,1 300μg,1 500μg,1 800μg)进行双向电泳,G-250考染后,通过PDQuest软件对双向电泳图谱进行比较分析。结果表明,三氯乙酸/丙酮法提取效果最好,所得单向SDS-PAGE条带数与2-DE蛋白点数均多于其它2种方法;主动水化对高丰度蛋白点聚焦效果较好;1 500μg蛋白上样量电泳图谱分辨率最高。该方法的建立为开展芒果蛋白质组学研究提供了参考。     

中枢神经蛋白质组分析中双向电泳技术的建立

建立和优化了中枢神经组织蛋白质组分析所需的双向电泳及相关技术.由于中枢神经组织结构的特殊性,样品处理非常困难.对样品液组成、样品处理、上样方式、上样量、IPG胶条和SDS-聚丙烯酰胺凝胶电泳染色方法和保存等相关技术进行了比较研究和条件优化后,以固相pH梯度等电聚焦为第一向和SDS均一胶（T=12.5%）的水平电泳为第二向,成功地得到了神经组织双向电泳图谱.     

Technical updates to basic proteins focalization using IPG strips

ABSTRACT: BACKGROUND: Gel-based proteomic is a popular and versatile method of global protein separation and quantification. However, separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility. RESULTS: Three different protocols of protein loading were compared using MCF7 cells proteins. In-gel rehydration, cup-loading and paper-bridge loading were first compared using 6--11 IPG strips, as attempted, in-gel rehydration gave large horizontal steaking; paper-bridge loading displayed an interesting spot resolution, but with a predominant loss of material; cup-loading was selected as the most relevant method, but still needing improvement. Twelve cup-loading protocols were compared with various strip rehydration, and cathodic wick solutions. Destreak appeared as better than DTT for strip rehydration; the use of isopropanol gave no improvement. The best 2DE separation was observed with cathodic wicks filled with rehydration solution complemented with DTT. Paper-bridge loading was finally analyzed using non-limited samples, such as bovine milk. In this case, new spots of basic milk proteins were observed, with or without paper wicks. CONCLUSION: According to this technical study of basic protein focalization with IPG strips, the cup-loading protocol clearly displayed the best resolution and reproducibility: strips were first rehydrated with standard solution, then proteins were cup-loaded with destreak reagent, and focalisation was performed with cathodic wicks filled with rehydration solution and DTT. Paper-bridge loading could be as well used, but preferentially with non-limited samples.     

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.     

An approach for identification of phosphoproteins using the G-electrode-loading method in two-dimensional gel electrophoresis

We assessed whether the G-electrode-loading method (GELM) was helpful in the protein analysis. GELM in 2-DE was compared with the slip-loading, the in-gel rehydration and the cup-loading in 2-DE. GELM showed the best results for protein separation. A total of 14 spots that showed an increase with GELM were analyzed by MALDI-TOF MS. In GELM, all of these spots were identified with a high score and a high sequence coverage. A membrane-associated protein was identified and determined to have phosphorylated site. These tests show that GELM has several advantages for protein analysis compared with the traditional methods.     

Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome

Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.     

Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels

The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5–5.5, 5–6, 5.5–6.7, 6–9 and 6–11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3–10 and 4–7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5–9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.     

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.     

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.     

Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis

Sample preparation is still the most critical step in two-dimensional gel electrophoresis (2-DE), and needs to be optimized for each type of sample. To analyze the proteome of the medicinal plant Catharanthus roseus, we developed and evaluated a sequential solubilization procedure for the solubilization of proteins after precipitation in trichloroacetic acid and acetone. The procedure includes solubilization with a conventional urea buffer followed by a stronger solubilizing buffer containing thiourea. The sequential solubilization of the precipitated proteins results in very different spot patterns following 2-DE. The number of protein spots which could be detected in both samples of the sequential solubilization was only about 10% of the total number of spots. Compared to solubilization in a single step, the total number of spots that could be detected in the sequential solubilization procedure was increased by 52%. The method described is simple and is applicable to different types of plant tissue.     

Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

Background

Trypanosoma cruzi, a flagellate protozoan, is the etiological agent of Chagas disease, a chronic illness that causes irreversible damage to heart and digestive tract in humans. Previous 2-DE analyses of T. cruzi proteome have not focused on basic proteins, possibly because of inherent difficulties for optimizing 2-DE in the alkaline pH range. However, T. cruzi wide pH range 2-DE gels have shown few visible spots in the alkaline region, indicating that the parasite either did not have an appreciable amount of alkaline proteins or that these proteins were underrepresented in the 2-DE gels.

Results

Different IEF conditions using 6–11 pH gradient strips were tested for separation of T. cruzi alkaline proteins. The optimized methodology described here was performed using anodic "paper bridge" sample loading supplemented by increased concentration of DTT and Triton X-100 on Multiphor II (GE Healthcare) equipment and an electrode pad embedded in DTT- containing solution near the cathode in order to avoid depletion of reducing agent during IEF. Landmark proteins were identified by peptide mass fingerprinting allowing the production of an epimastigote 2-DE map. Most identified proteins corresponded to metabolic enzymes, especially those related to amino acid metabolism. The optimized 2-DE protocol was applied in combination with the "two-in-one gel" method to verify the relative expression of the identified proteins between samples from epimastigote and trypomastigote life stages.

Conclusion

High resolution 2-DE gels of T. cruzi life forms were achieved using the optimized methodology and a partial epimastigote alkaline 2-DE map was built. Among 700 protein spots detected, 422 were alkaline with a pI above 7.0. The "two-in-one gel" method simplified the comparative analysis between T. cruzi life stages since it minimized variations in spot migration and silver-stained spot volumes. The comparative data were in agreement with biological traits of T. cruzi life forms and also corroborated previous T. cruzi proteomic studies. For instance, enzymes related to amino acid metabolism and dehydrogenases were more abundant in epimastigote 2-DE gel whilst trans-sialidase and a paraflagellar protein were found specifically in the trypomastigote 2-DE profile.     

多子房小麦蛋白质双向电泳体系的建立及优化

为建立适用于显性多子房小麦细胞质效应的蛋白质双向电泳体系,以显性多子房小麦材料DUOII与特异细胞质材料TeZhiI杂交的F1幼穗为材料,采用TCA-丙酮法提取蛋白质,并在IPG胶条长度和pH范围、SDS-PAGE凝胶浓度及蛋白质上样量等方面,对多子房小麦幼穗蛋白质双向电泳体系进行了探究与优化.结果表明,本文采用的蛋白质定量方法准确度高(R²=0.9999),确立了17 cm, pH4～7的IPG胶条, 12% SDS-PAGE分离胶,上样量为900 μg的双向电泳方法体系,获得了最适合本研究蛋白质组分析的双向电泳图谱. 经PDQuest 2DE 8.0.1软件分析,2-DE图谱上可分辨出1.444±14个清晰蛋白质点,且重复性较高(95%), 相关系数为0.960. 建立了一套适用于显性多子房小麦细胞质效应研究的蛋白质双向电泳体系.