Name: multimer-page: a method for capturing and resolving protein complexes in biological samples
Uploaded: 2017-05-05T14:00:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

由NIH / NIDA DA034783支持。我们感谢布赖恩·A·基兰热与多聚-PAGE技术援助。

1.组织制备<ol><li>制备4×BN-PAGE样品缓冲液（200mM双（2-羟乙基）氨基 - 三（羟甲基）甲烷（的Bis-Tris），200mM的氯化钠，40％w / v的甘油，0.004％丽春红S，pH为7.2的10毫升）。 注意:此储备溶液可以预先制成，并储存在4℃。 </li><li>稀释4倍的样品缓冲液的250μL750μL卫生署2含有1x商业蛋白酶抑制剂混合物O操作。涡和冰中冷却。 </li><li>均质化20毫克目标组织的在用干净的Dounce匀浆的30个行程1个毫升1X冰冷BN-PAGE样品缓冲液中。 注:对于这个演示实验中，目标组织是整个大鼠脑组织。均化后，样品可以用温和的去污剂如2％毛地黄皂苷处理以溶解和允许膜蛋白的电泳。 </li><li>转移匀浆到1.5mL微量离心管中，并离心以14,000×g离心30分钟以沉淀不溶细胞内容物。 AFTEř离心，倒出上清液到在冰上干净的管中。 </li><li>遵循制造商的说明书使用二辛可宁酸（BCA）或类似的蛋白定量测定法来测量上清液蛋白质浓度。 </li><li>如果匀浆样品（一个或多个）含有的洗涤剂，在水溶液中足以使匀浆溶液至0.25％考马斯添加5％考马斯蓝G-250的量。 </li></ol> 2. BN-PAGE <ol><li> 稀释25毫升20×蓝色天然（BN）电泳缓冲液的库存（1.0M的Bis-Tris，1.0M甘氨酸，pH6.8）中为475毫升含卫生署0.002％考马斯蓝，使500毫升1×电泳缓冲液的2 O。 <ol><li>如果样品含有洗涤剂，代替使250毫升含0.02％考马斯1X运行缓冲液和含有无另一个250毫升。 </li></ol></li><li>冷硬缓冲器（一个或多个），以4℃。 </li><li>清洗和组装的聚丙烯酰胺凝胶，根据制造商的说明书浇注盒。 </li><li&gt;根据标准配方7倾BN的聚丙烯酰胺凝胶（3％T堆叠层，6％T分解层），并放置好梳子。 </li><li>凝胶具有聚合后，冲洗与卫生署2 O的盒子和根据制造商说明书组装电泳装置。 </li><li> 删除以及梳子，并与1X BN-PAGE电泳缓冲液填充井。避免用缓冲液填充整个内室，直到样品被加载。 <ol><li>如果样品含有洗涤剂，填孔用含有0.02％考马斯蓝的缓冲区。 注意:在4℃下执行步骤2.7-2.9。 </li></ol></li><li>使用凝胶加载提示，吸取含有20μg蛋白质的成所需井匀浆的体积。移液器1X BN-PAGE样品缓冲液的等体积到任何未使用的孔中。 注意:使用从BCA测定法测定蛋白质浓度，以计算匀浆适当体积添加到井。 </li><li> 样品装载之后，填充用1x BN-PAGE电泳缓冲液内室。确保凝胶在缓冲液完全淹没。接着，填充用1x运行缓冲液外室由制造商所指示的电平。 <ol><li>如果样品含有洗涤剂，填用含有0.02％考马斯蓝的1X缓冲液，并用无染料1X运行缓冲液外室的内室。 </li></ol></li><li>将电极连接到电源，并在150V electrophorese将凝胶中的蛋白质直到染料带前进〜2厘米的分辨层。停止并断开电源。 </li></ol> 3.交联注意:在4℃下执行步骤3.1-3.6。 <ol><li>拆卸电泳装置中，并分离凝胶盒的玻璃板。卸下并丢弃堆叠层。 </li><li>小心切开刚好低于染料前端的底部边缘的凝胶。采取护理使这种馏分作为平直越好，然后丢弃未使用的片凝胶。这将留下〜2厘米宽，聚丙烯酰胺凝胶的水平条，其中将包含匀浆样品中的所有蛋白质。 </li><li>修剪掉沿凝胶条带的边缘的任何未使用的部分。 </li><li>小心地将条插入在一个小容器10毫升磷酸盐缓冲盐水（PBS），并轻轻地由章动30分钟以平衡混合。 </li><li>平衡之后，丢弃并用另一个10毫升替换PBS。吸移管500的μL的25mM DSP溶解于二甲基亚砜入PBS，并继续30分钟如上（步骤3.4）混合。 </li><li>倒掉DSP溶液。添加0.375的1M Tris（羟甲基）氨基甲烷盐酸盐（的Tris-HCl），pH值8.8，含有2％十二烷基硫酸钠（SDS）10毫升以猝灭未反应的DSP。继续垂头15分钟。 </li></ol> 4. SDS-PAGE <ol><li>虽然凝胶条带淬火，制备SDS-PA根据标准方法9 GE凝胶溶液。不添加聚合试剂。 </li><li> 骤冷后，返回ΒΝ凝胶条带至室温，并在带材浇注到一个新的凝胶盒。 <ol><li>要做到这一点，小心拿起凝胶，并将其放置到一个干净的凝胶盒隔板。 </li><li>东方带所以染料前端的底部最接近新磁带盒的顶部（ 即 ，在BN-PAGE行进最远的一侧应该被最接近新磁带盒的顶部;将凝胶条应从其先前被翻转方向）。参见图3。 </li><li>放置条带，使得其顶部边缘在于即使其中所述盖板的顶部边缘会（ 即 ，这将是在新的凝胶的顶部）。确保染料前端平行于所述玻璃板的水平边缘。 </li><li>推压隔板壁中的一个切下的带材的一侧上，留下上吨室他另一侧用于凝胶被倾并且要加载的蛋白质标准或梯子。 </li><li>如果凝胶条的底部边缘中包含任何锯齿或不平整区域，小心切割他们离开。如果存在的话，它们将凝胶中的气泡陷阱浇注。 </li><li>一旦凝胶条被正确地定位，笼罩间隔板盖板。轻轻推压，推出任何夹带的气泡。 </li><li>继续根据制造商说明书来组装凝胶浇注装置。 </li></ol></li><li>添加聚合试剂分离胶缓冲液，并且使用血清移液管倒在制得的凝胶盒，。填充凝胶盒至〜2厘米切除BN-PAGE凝胶带的下方，以留有余地，堆叠层。 </li><li>添加100μL丁醇在倾倒凝胶的顶部，并允许分辨层的聚合30分钟。倒掉丁醇。 </li><li> 添加聚合试剂于堆叠凝胶溶液。使用serolog移液管的iCal，倒入堆叠层以填充在凝胶盒的所有剩余空白空间。 <ol><li>倾斜凝胶盒作为层叠层倾倒，使其填充胶条下方的空间，并且气泡不会夹。 </li><li>作为层叠凝胶缓冲液填充该凝胶条下面的空的空间，逐渐将凝胶盒返回到电平的基础上。 </li><li>不断填补空白与浓缩胶缓冲切除的胶条旁边，直到它几乎溢出。 </li></ol></li><li>允许堆叠层聚合30分钟。 注意:请10X SDS-PAGE电泳缓冲液（250mM的三（羟甲基）氨基甲烷（TRIS），1.9甘氨酸，1％SDS），同时将凝胶聚合。这也可以预先制成，在室温下储存。 </li><li>稀释50毫升10X SDS-PAGE电泳缓冲液库存入450毫升卫生署2 O，使1×操作缓冲液。 </li><li>堆叠层具有聚合后，从浇注取出凝胶盒设备，冲洗与卫生署2 O，并根据制造商说明书组装电泳装置。 </li><li>用1x SDS-PAGE电泳缓冲液完全充满内腔，然后填充该外室由制造商所指示的电平。 </li><li>装入空间的分子量梯或适当蛋白质标准的切下的凝胶条带旁边。 </li><li>连接电极到电源，并且当考马斯染料跑出凝胶electrophorese在120 V.样品，停止和断开电源。 </li><li>分析使用电印迹和蛋白质检测的标准方法通过抗体结合10的凝胶。 </li></ol>

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蛋白质 - 蛋白质相互作用是每个任务万物开展重要。正因为如此，他们是严格审查和研究的课题。多聚-PAGE是用于捕获，分离，和分析广泛的蛋白复合物的新方法。之前我们已经证实其适用于研究疾病相关蛋白α突触核蛋白11 oligimerization。然而，它是可扩展到许多蛋白复合物，如在图2证实。当与学习蛋白复合物的其他方法，多聚-PAGE是因为它的简单和简洁的是有利的?...

<html>正常细胞功能是依赖于蛋白质-蛋白质相互作用1,2。其结果是，人类疾病通常是由在不同的蛋白质复合物3的组件和行为的扰动标记。表征这种相互作用的能力因此，至关重要。检测这些相互作用电流的装置需要的靶蛋白，通常随后通过相互作用配偶的下拉的纯化。常规纯化通过差速离心，沉淀和/或色谱法4来实现的。这些方法是耗时的，必须改变用于每个靶蛋白，并经常导致低的产率。现代纯化方法涉及肽标签融合到靶蛋白，随后通过免疫沉淀或萃取上装载有结合于捕获分子5珠粒柱，"&gt; 6，虽然这是可扩展到许多蛋白质，这需要目标的序列修饰，潜在地导致在亲和力差异，它们通常的结合配偶体的构建体。一些蛋白质-蛋白质相互作用的微妙特性意味着这种方法可能不适用许多方案。另外，用于映射蛋白质 - 蛋白质相互作用的下拉测定法可以不捕获准确蜂窝图片，由于受限制的自由度和诱饵蛋白的非天然的水平。 理想情况下，蛋白复合物能够在他们的原生状态进行检测，而不需要净化或下拉。蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）被开发作为变性少替代十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），并允许某些蛋白质和复合物从生物样品7的分离。然而，在BN-PAGE蛋白质分离的基础上LARGE数的变量，包括大小，电荷，三维结构，和关联与其他分子。这些因素的相互作用通常导致蛋白质的共分离，聚集体形成，差的蛋白质条带的分辨率。二维原生聚丙烯酰胺凝胶电泳解决了一些，但不是全部，这些问题8。 为了避开与天然分离相关的并发症，一些作者使用胺反应性的交联试剂，如二硫代双（琥珀酰亚胺丙酸酯）（DSP），以捕获蛋白复合物在组织裂解物4。然后将这些处理过的溶胞产物可变性，并通过SDS-PAGE分离，同时保持蛋白质复合物的天然大小和化妆。然而，由于交联试剂反应基于一个分子到另一个的接近度，以及在组织裂解液的蛋白质具有许多自由度，并且可以随机相互作用，非特异性背景交叉-linking可以是高的，尤其是浓缩的样品英寸这可能会导致难以解释结果。 在这里，我们证明了使用混合BN-PAGE / SDS-PAGE的方法，被称为多聚聚丙烯酰胺凝胶电泳（多聚-PAGE），以分离和复杂混合物中检测蛋白组件。最初，细胞裂解物通过BN-PAGE悬浮于聚丙烯酰胺凝胶。含溶胞产物 - 凝胶，然后用交联试剂DSP反应。伪固定，并略微在凝胶上分离，蛋白可能非特异性反应少得多，这意味着背景交联剂反应性降低。交联后，将凝胶条带切下并通过SDS-PAGE分离。将所得的凝胶然后可以通过典型地与聚丙烯酰胺凝胶电泳相关的任何手段进行分析。这种方法允许在未修饰的组织裂解物天然蛋白质复合物的分离和检测，而无需额外的纯化或上拉陶氏ñ。

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;epsilon;-Aminocaproic acid</td><td>Sigma</td><td>A2504</td><td></td></tr><tr><td>Acrylamide</td><td>Acros Organics</td><td>164855000</td><td>Toxic.</td></tr><tr><td>Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)</td><td>BioRad</td><td>161-0148</td><td>Toxic.</td></tr><tr><td>Ammonium persulfate</td><td>Sigma</td><td>A3678</td><td></td></tr><tr><td>Anti rabbit IgG-HRP from goat</td><td>Santa Cruz Biotechnology</td><td>sc-2004</td><td></td></tr><tr><td>Bicinchoninic acid assay kit</td><td>Thermofisher</td><td>23225</td><td></td></tr><tr><td>Bis-tris</td><td>Sigma</td><td>B9754</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Butanol</td><td>Fisher Scientific</td><td>A399-1</td><td></td></tr><tr><td>Chemiluminescence substrate kit</td><td>ThermoFisher</td><td>24078</td><td></td></tr><tr><td>Coomassie blue G-250</td><td>Sigma-Aldrich</td><td>B0770</td><td></td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Toxic.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher Scientific</td><td>D128-1</td><td></td></tr><tr><td>Dithiobis(succinimidylpropionate)</td><td>Thermo Scientific</td><td>22585</td><td></td></tr><tr><td>Dry nonfat milk</td><td>LabScientific</td><td>M0841</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>Thermofisher</td><td>78430</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144SI-212</td><td>For titration. Caustic.</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412-4</td><td>For PVDF membrane activation. Toxic.</td></tr><tr><td>Monoclonal anti &amp;alpha;-synuclein IgG from rabbit</td><td>Santa Cruz Biotechnology</td><td>sc-7011-R</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-tetramethylethylenediamine</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>N,N&#039;-methylenebisacrylamide</td><td>Acros Organics</td><td>16479</td><td>Toxic.</td></tr><tr><td>NP40</td><td>Boston Bioproducts</td><td>P-872</td><td></td></tr><tr><td>Polysorbate 20 (tween-20)</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Polyvinylidene fluoride transfer membranes</td><td>Thermo Scientific</td><td>88518</td><td></td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P3504</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P217-3</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Thermo Scientific</td><td>88265</td><td></td></tr><tr><td>SDS solution (10% w/v)</td><td>BioRad</td><td>161-0416</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma</td><td>L37771</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Fisher Scientific</td><td>BP329-1</td><td></td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-500</td><td></td></tr><tr><td>Tris-HCl (0.5 M buffer, pH 6.8)</td><td>BioRad</td><td>161-0799</td><td></td></tr><tr><td>Tris-HCl (1.5 M buffer, pH 8.8)</td><td>BioRad</td><td>161-0798</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE Imagequant LAS 4000</td><td>GE Healthcare</td><td>28-9558-10</td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td></td><td></td></tr><tr><td>Synergy H1 microplate reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Gel Former + Stand</td><td>Biorad</td><td></td><td></td></tr><tr><td>Microfuge 22R centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>2 mL dounce homogenizer</td><td></td><td></td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Ultrasonic tissue homogenizer</td><td>Fisher Scientific</td><td>FB120220</td><td></td></tr></tbody></table>

multimer-page: a method for capturing and resolving protein complexes in biological samples

有许多用于净化和研究单个蛋白质和肽发达的方法。然而，大多数的细胞功能是由相互作用的蛋白复合物的网络，这往往是难以调查，因为它们的结合是非共价的，并通过纯化技术容易扰动进行。这项工作描述了稳定剂和使用二维聚丙烯酰胺凝胶电泳从未经修饰的组织分离天然蛋白质复合物的方法。组织裂解液加载到非变性蓝色天然聚丙烯酰胺凝胶，然后施加电电流，直到蛋白质迁移的短距离到凝胶。然后将含有已迁移蛋白质凝胶条带切出，并与该胺反应性交联剂二硫代双（丙酸琥珀酰亚胺酯），其共价稳定的蛋白复合物一起温育。然后将含有交联复合物的凝胶条带浇注成十二烷基硫酸钠聚丙烯酰胺凝胶，和叔他配合是完全分开的。该方法依赖于技术和最熟悉的分子生物学家的材料，这意味着它是价格低廉，简单易学。虽然在其充分分离非常大的复合能力是有限的，并没有受到普遍成功，该方法能够捕捉各种充分研究的复合物，并有可能适用于许多感兴趣的系统。

在这个演示实验，多聚-PAGE是在整个大鼠脑裂解液进行。将所得的分离的蛋白质印迹到聚偏二氟乙烯（PVDF）膜上，然后用针对已知能形成复合物的蛋白质的抗体进行探测。 图1示出了通过两种方式的协议的验证。首先，我们表明，交联的蛋白是可裂解通过加入还原剂的，这意味着所观察到的较高分子量的物质通过交联试剂形成，并且不是由于协议（ 图1A?...

Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

一种用于稳定和使用胺反应性蛋白交联剂偶联到一种新颖的二维聚丙烯酰胺凝胶电泳分离未修饰的组织裂解物天然蛋白质复合物的方法（PAGE）系统被呈现。

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

multimer-page-method-for-capturing-resolving-protein-complexes

Research

JoVE Journal

Biochemistry

11.3K 次观看.  Michigan State University. 一种用于稳定和使用胺反应性蛋白交联剂偶联到一种新颖的二维聚丙烯酰胺凝胶电泳分离未修饰的组织裂解物天然蛋白质复合物的方法（PAGE）系统被呈现。 

视频: Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

生物学

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

发现蛋白质相互作用和检定蛋白质功能使用的HaloTag技术

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...