蛋白质电泳-在蛋白质组学中的应用.doc

原 理 蛋白质分子在溶液中由于其末端氨基、末端羧基及侧链的游离基团而成为带电颗粒，并可在电场内移动，其移动方向取决于蛋白质分子所带静电荷。不同蛋白质分子根据其氨基酸组成及所在溶液的pH值，携带的静电荷不尽相同，致使它们在电场中的迁移率各异，从而达到分理的目的。电泳原理示意图在蛋白质组学中对电泳的分类一维电泳(one dimensional gel electrophoresis, 1DE)，现在普遍采用垂直板聚丙烯酰胺凝胶电泳（PAGE）二维电泳(two-dimensional gel electrophoresis, 2DE)，又称双向电泳一维电泳现在普遍采用聚丙烯酰胺凝胶电泳（PAGE），包括非变性电泳(native PAGE)和SDS-PAGE两种，前者主要是在分离蛋白复合物时经常用到。后者是在凝胶与缓冲系统中加入阴离子表面活性剂十二烷基硫酸钠（SDS），蛋 白质分子被大量SDS阴离子包裹，消除了它们间原来携带的电荷差别，因而其迁移率仅反映蛋白质分子大小，故广泛用于蛋白质分子量的测定。凝胶浓度和蛋白质分离范围缓冲液的选择通常在SDS-PAGE均选择Tris-glycine作为电泳的缓冲系统。但在大部分缓冲系统中，SDS微团 (micelle)会干扰小分子蛋白质的分离，而Tris-tricine系统则可使小蛋白质SDS复合物与微团分离，去除干扰。此外，也证明该系统对 脂多糖和脂寡糖混合物的分离有效。12%胶常用的低分子量标准蛋白名称来源分子量磷酸化酶B兔肌肉97400牛血清白蛋白牛66200卵清蛋白鸡蛋清45000碳酸酐酶牛31000胰蛋白酶抑制剂大豆21500溶菌酶鸡蛋清14400一维凝胶染色现在用于凝胶中蛋白染色的方法包括氨基黑10、考马斯亮蓝R250、考马斯亮蓝R350、银染、铜染及橙染(sypro orange)。最常用的为考马斯亮蓝R250、考马斯亮蓝R350染色，为了提高灵明度采用银染。一维电泳的应用初步测定蛋白质的分子量初步分离蛋白质复合物，并进一步用于免疫组化分析对重组表达蛋白的初步鉴定将一维电泳和生物质谱相结合，达到对较简单蛋白复合物的分离和鉴定一维电泳在蛋白质组研究中的应用举例我们将一维电泳和LC-Ms/MS结合，成功进行如下研究： (1)血浆蛋白质组研究 (2)SARS病毒蛋白的分析鉴定,成功鉴定了SARS病毒的S蛋白、N蛋白和E蛋白，有力推动了我国第一个SARS疫苗的研究和申报工作。双向电泳(2DE)原理：2DE就是第一向采用等电聚焦分离，第二向为SDS-PAGE分离。由于 2DE是依据蛋白质的两种不同性质，即等电点和分子量进行分析，因此分辨率远高于其他任何一种单一的电泳方法，是目前分析混合蛋白质样品最有效的手段。 （注意：双向电泳中的“双向”指的是按照蛋白质的两个性质即“等电点和分子量”进行分离的原理。)2DE仪器系统2DE在蛋白质组学方案中所处的位置这就是将2DE和质谱相结合进行比较蛋白质组分析的实验方案。（方案完后），下面，我将汇报用以上介绍的方法进行脾淋巴细胞及乳腺癌比较蛋白质组学的实验结果。双向电泳的操作程序(以进行差别表达蛋白组分析为例)1. 样品制备2. IPG strips重泡胀及上样(rehydration and liading)3. 等电聚焦4. 胶条平衡，包括还原和烷基化5. 第一向胶条转移到第二向6. SDS-PAGE7. 染色8. 图像分析目前，普遍采用预制的固定pH梯度胶条(immobilized pH gradient strips, IPG strips)。商品化的IPG strips可以从Amersham Pharmacia公司或BioRad公司购买。IPG胶条制备(casting of IPG strips)IPG slab gels with linear gradients pH 4-7, 4-9, 6-10 and 4-12 are cast according to Grg et al. (1986) with the recipes of Righetti (1990) and Grg et al. (1998). Two starter solutions (an acidic one and a basic one) are prepared as described in Table 1. For better polymerization, the acidic and basic solutions are adjusted to pH 7 with sodium hydroxide and acetic acid, respectively. ProcedureTo assemble the polymerisation cassette wet the plain glass plate (size 260 x 200 mm2) with a few drops of water. Place the Gelbond PAGfilm, hydrophilic side upwards, on the wetted surface of the plain glass plate. The GelBond PAGfilm should overlap the upper edge of the glass plate for 1-2 mm to facilitate filling of the cassette. Expel ecxess water with a roller. Place the glass plate which bears the U-frame (0.5 mm thick) on top of the GelBond PAGfilm and clamp the cassette together. Put it in the refrigerator for 30 min. Assembly of the gel casting cassette (Left): Assembly of the polymerisation cassette for IPG and SDS gel casting on plastic backing (Glass plates, GelBond PAGfilm, U-frame 0.5 mm thick)(Right): Application of the GelBondPAGfilm onto the glass plateIPG gel casting (1) Gradient mixer and connecting valve; (2) outlet tubing valve; (3) gel casting cassette The acidic, dense solution is pipetted into the mixing chamber and the basic, light solution into the reservoir of the gradient mixer, an extra portion of the dense solution is prepared and pipetted into the mold prior to pouring the gradient. After pouring the gradient into the precooled mold (refrigerator), the mold is kept at room temperature for 15 min to allow adequate levelling of the density gradient prior to polymerization for one hour at 50C. After polymerization, the mold is kept at room temperature for at least 15 min. Then the IPG gel is removed from the mold and extensively washed with deionized water, impregnated with 2% glycerol, and dried at room temperature in a dust-free cabinet and, if not used immediately, covered with a plastic film for storage at -20C. The dried gels can be stored frozen for at least one year. Note: In order to ensure the reproducibility of the IPG gradient, the volume of the cassette should be constant. Therefore, it is recommended to check the volume of the cassette from time to time since it diminishes on ageing of the U-frame.Note: When one of the chambers is emptying faster than the other, the resulting pH gradient will not be linear. Check if there is an air bubble in the connecting line or whether the speed of the magnetic stirrer is not appropriate!1. 样品制备样品裂解液(lysis solution): 8M urea, 4% CHAPS, 40 mM Tris base, 65 mMDTE 制备后的裂解液分装冻存20备用。 如果必要，urea的浓度可增加到9或9.8M。 Triton X-100, NP-40及其他的非离子型去污剂或Zwitterionic detergents可取代CHAPS对于脂蛋白，膜蛋白的分析，可以用如下的蛋白质裂解液：7M urea, 2M thiourea, 4% CHAPS, 40 mM Tris base, 65 mMDTEPresulubilization of protein in (boiling) SDS buffer, followed by dilution with urea lysis buffer.Initially solubilized in 0.5-1% SDS,followed by dilution with at least an eight excess of 2-4% (w/v) NP40, Triton X-100, or CHAPS to reduce the final concentration to 0.25%. This dilution displaces the SDS from the proteins and replaces it with a nonionic or zwitterionic detergent.制备2DE样品时常见的污染物盐、小离子性分子(small ionic molecules)、离子型去污剂(ionic detergent)、核酸、多糖、脂类以及酚类化合物等，这些物质会直接影响2DE的效果。脂的去除 Lipids may interact with membrane proteins and consume detergents.High speed centrifugation of lipid-rich materialExtraction of the biological material with organic solvent (e.g.,ethanol or acetone)However, loss of proteins because certain proteins are soluble in the organic solvent or because the precipitated proteins do not always resolubilize核酸的去除 Nucleic acids can interact with carrier ampholytes and proteins and give rise to 2D patterns containing horizontal streaks and increase the viscosity of the solution and may clog the pores of the gels.TCA/aceton (typically, 20%TCA in pure acetone) precipitation;Protease-free RNase and DNaseUltracentrifugation and addition of a basic polyamine such as spermine;sonication制备样品的原则样品制备步骤越简单越好，避免目的蛋白的损失或丢失。裂解细胞或组织要防止蛋白降解。裂解细胞应在低温下进行(冰水浴或4)，最好用加有蛋白酶抑制剂的裂解液直接裂解。样品裂解液应新鲜配制，或分装冻存，而且要采用高纯度的去离子尿素蛋白样品应在等电聚焦前新鲜配制，或-80分装冻存，绝对避免将样品反复冻融。通过超离心去除所有颗粒性物质，因为颗粒性物质或脂会阻塞凝胶孔路。为了防止蛋白质被修饰，加入尿素后，不要加热蛋白样品。当样品中含有尿素时，加热绝对不要超过37.升高温度会使尿素水解成异氰酸盐(isocyanate),使蛋白发生氨甲酰化(carbamylation)修饰。最好用强烈变性剂直接裂解样品，这样可以使蛋白水解酶立即变性失活。细胞裂解方法比较温和的裂解方法包括：渗透裂解(Osmotic lysis)，此法特别适合分离亚细胞组分，如血细胞、组织培养细胞的裂解；冻融裂解法(Freeze-thraw lysis)，快速将细胞冻于液氮中，然后融解，重复进行，如细菌、组织培养细胞的裂解；去污剂裂解法(detergent lysis)，去污剂融解细胞膜，从而裂解细胞，释放其中成分，如组织培养细胞的裂解；酶裂解(enzymatic lysis)，具有细胞壁的细胞通过酶除去细胞壁。细胞用溶菌酶(lysozyme)，植物细胞用纤维素酶(cellulase), 果胶酶(pectinase),酵母用溶细胞酶(lyticase)。强烈的裂解方法包括：超声波破碎，但要注意减少热能和泡沫的产生，以防止蛋白质变性或被剪切。手工研磨，常可以加入石英砂。或采用研钵(mortar)，或匀浆器(pestle)在液氮中研磨，如固体组织或微有机体的研磨。蛋白酶抑制剂苯甲基磺酰氟(PMSF, 1mM)，PMSF为酶的不可逆抑制剂，用于serine proteases及cysteine proteases的抑制。1mM EDTA或1mMEGTAPeptide protease inhibitorsAEBSF (4mM)苯脒(Benzamidine) (1-3mM)其中，PMSF,EDTA及肽蛋白酶抑制剂, e.g., leupeptin(亮抑酶肽), pepstatin(胃蛋白酶抑制剂), aprotinin(抑制丝氨酸蛋白酶的碱性多肽), bestatin(亮氨酸类似物）是最常用的几种蛋白酶抑制剂。常用的蛋白质沉淀方法硫酸铵三氯乙酸(TCA)丙酮，放于-20时效果会更好TCA丙酮2. IPG 胶条重泡胀(rehydration)和上样(loading)因为采用预制的IPG干胶条，需要用重泡胀液(rehydration solution)使其重性水化膨胀成凝胶，使样品能够充分进入凝胶内，完成上样。 可用重泡胀液在室温浸泡12h后上样，或在低电压(30v)下室温12h,使IPG干胶条边水化膨胀边上样。Rehydration solution8 M urea, 4% CHAPS, 100 mM DTT, 0.5-2% IPG buffer/pharmalyte3. 第一向 等电聚焦(isoelectric focusing, IEF)IPG Phor等电聚焦仪典型的IEF条件举例采用安玛西亚公司IPG Phor等电聚焦仪，pH 3-10的非线性胶条，蛋白上样量250g，体积350l。电流为50Astrip，所用电泳条件为30V,12h;100v,1h; 500v, 1h;1000v,1h; 8000v,1h(gradient); 8000v,10h，共用时26h，IEF结束后vhs为80000左右。4. IEF后胶条的平衡 SDS平衡缓冲液为：50mM Tris-Cl pH8.8, 6M Urea, 30% glycerol, 2% SDS, trace bromophenol 还原：100mg DTT/10 ml SDS 平衡buffer，15min; 烷基化：250mg iodoacetamide/10ml SDS平衡buffer, 15min. 5. 胶条向第二向转移将IEF后的胶条转移到垂直板聚丙烯酰胺上，用1%的琼脂糖封闭，从而使胶条上的蛋白转移到第二向分离胶中，根据分子量大小将pI接近的蛋白进行进一步分离。6. SDS-PAGE注意：进行第二向SDS-PAGE时，要求恒温，但温度不要低于15，否则会引起凝胶收缩，影响蛋白从胶条向第二向凝胶的转移。7. 2D胶的染色考马斯亮蓝染色，常用考马斯亮蓝R250,R350(sensitivity: 1ug/spot)负染(negative staining), 如锌染 (ST: 50 ng)银染，为了有利于对2D胶上的蛋白进行质谱鉴定，最好不要使用戊二醛，因为戊二醛会和蛋白质交联，降低肽提取效率(0.1 ng)。荧光染色，为了提高灵敏度，常采用荧光染料染色,e.g., SYPRO Ruby, ST:1-2 ng, single step, available for automation放射性同位素染色(125I,32P, 14C or 35S),标记的蛋白质直接进行放射自显影Comparison of fluorescent labelled 2-D gel with silver staining8. 图像分析在用双向电泳进行差别表达蛋白质组分析时，需要利用软件对产生的2D胶进行差别分析，以寻找差别表达蛋白。常用的软件如安玛西亚公司的Image Master 2D Elite或Image Master 2D Planium分析软件。图像分析一般程序Image visualization and manipulationSpot detection and quantificationSpot matchingEditing matchesMolecular weight and pI calibrationSynthetic and average gelsAnalyzing the dataAnnotating the gelsImage analysis with ImageMaster PlatinumImage analysis with ImageMaster PlatinumSpots report差别分析(Differential analysis)表达增高的点（Up-regulated spots）表达降低的点（Down-regulated spots）只在样品或对照品中出现的特异性点差别分析示意图表达上调表达下调的点(right)2DE常见问题及解决办法(Troubleshooting)Too much salt in the sample (disturbs IEF)Hint:Include an acetone precipitation to remove salts and other contaminants Charged impurities in the sampleHint:Include an acetone precipitation to remove salts and other contaminantsImpurities in the sample or rehydration solutionHint:Include an acetone precipitation to remove salts and other contaminantsPrepare new rehydration solution with pure reagents underfocused (Focusing time not long enough)Hint:Prolong the focusing time of the IEF Gel surface during polymerization not overlaid with destilled water (or too low amount of water used)Hint:Apply at least 1 ml to overlay the gel surface No uniform gel polymerization (e.g. impurities at gel cassettes, air bubbles in the polymerized gel)Hint:Clean gel cassettes with ethanolCast the slab gel slowly (approx. 1 min)Overlay the gel carefully with destilled water Not all proteins (especially high molecular mass proteins) saturated with SDSHint:Use 0.15 % instead of 0.1 % (w/ v) SDS in the 1 x SDS buffer for SDS-PAGE High sample load (protein disturbs separation of other spots or may not be fully saturated with SDS)Hint:Lower the protein amountImpurities on or within the 2-D gel still present during silver stainingHint:Clean the gel cassettes prior casting with ethanolIncubate the 2-D gels long enough (and with at least 100 ml/ gel) in fixing and washing solution prior staining 进行2DE分析几点建议样品制备是关键，务必使蛋白样品充分裂解，除去颗粒性物质，如核酸、糖，盐及去污剂等的影响要注意蛋白从第一向到第二向的转移，可用分离胶直接封闭IPG胶条为了提高银染的效果，玻璃板和染色盘一定要清洗干净如要进行差别蛋白组分析，银染的方法要注意和质谱分析相匹配提高2DE分辨率的策略首先采用宽pH范围的胶条，确定蛋白的分布范围，通常采用pH3-10的胶条如果pH线性分布的胶条分离效果不好，可采用非线性胶条加大蛋白上样量，提高局部蛋白的分辨率采用窄pH范围的胶条，提高某pH范围蛋白的分辨率第二向采用梯度胶进行分离去除高丰度蛋白，进行蛋白的fractionation处理,富集低丰度蛋白采用窄范围pH胶条对pH4-7的2D胶进行局部分辨率提高的分析举例pH4-7胶条分析的2D胶图谱pH4-5胶条分析的2D胶图谱pH4-5胶条分析的2D胶图谱pH5.5-6.7胶条分析的2D胶图谱如上所示：通过分别用pH4-5,pH4-6,pH5.5-6.7的窄pH范围胶条，较单独用pH4-7的胶条，可大大提高局部区域的分辨率，使检