d.r. C 是一个癌症研究所新南威尔士的研究员和 d.n. S 以前是一个癌症研究所新南威尔士州研究员。本手稿中的研究结果由新南威尔士癌症研究所 (13/FRL/1-02 和 09/民防部队/2-39)、澳洲 (项目赠款 GNT1052963)、爱尔兰科学基金会 (11/SIRG/B2157)、新南威尔士州科学和医学研究办公室、来宾家庭资助。奖学金和 Mostyn 家庭基金会。J.F.H. 和 R.S. 是澳大利亚研究生奖获得者。

1. 质粒克隆
注意: 要生成质粒载体, 将 V1 或 V2 标记融合到感兴趣基因的 N 终点或 C 终点, BiFC 的目标向量已存入 Addgene [N 末端标记: pDEST-V1-ORF (#73635), pDEST-V2-ORF (#73636)。C 终端标签: pDEST-ORF-V1 (#73637), pDEST-ORF-V2 (#73638)]。该基因/s 的兴趣将需要在特定的重组克隆兼容进入媒介 (即pDONR223 或 pENTR221), 没有停止密码子, 以进行克隆。许多相容的克隆已经在各种质粒集合中获得, 包括哺乳动物基因组收集 (https://genecollections.nci.nih.gov/MGC/)。
<ol><li>执行重组反应, 在适当的 BiFC 目标向量的 attR 站点之间插入感兴趣的基因:<ol>
<li>在0.2 毫升管中, 添加 150 ng 进入向量, 150 ng BiFC 目标向量, 8 µL TE 缓冲器 (pH 8.0)。</li>
		<li>添加2µL recombinase 酶组合 (见材料表)。搅拌好, 离心机简单。</li>
		<li>在室温或16摄氏度的一夜内孵化1小时的反应。</li>
		<li>通过添加1µL 蛋白酶 K 溶液和孵化37°c 10 分钟来停止反应。</li>
	</ol>
</li>
	<li>用 LR 反应产物转化热休克主管细胞 (见材料表): 注: 任何基本菌株都可以在这一点上使用, 只要它不含有 Ccdb 抗毒素, 这将防止特定的选择的质粒含有插入。<ol>
<li>解冻有能力的细胞在冰和转移50µL 细胞成14毫升圆底聚丙烯管。</li>
		<li>在细胞中加入1µL 的反应产物, 并轻轻地混合。在冰上孵化20分钟。</li>
		<li>热休克在42°c 水浴四十五年代. 立即返回冰2分钟。</li>
		<li>添加1毫升的 LB 培养基和孵育37°c 1 小时。</li>
		<li>将其转化为含有氨苄西林 (100 µg/毫升) 的10厘米琼脂板, 并在37摄氏度一夜孵育。</li>
	</ol>
</li>
	<li>BiFC 质粒的纯化。<ol>
<li>要从单个菌落中纯化质粒, 将100毫升的 LB 介质添加到大锥形瓶中, 加入氨苄西林 (100 µg/毫升)。要筛选多个菌落, 请在14毫升圆底聚丙烯管中添加5毫升的 LB 介质, 并加入氨苄西林 (100 µg/毫升)。</li>
		<li>使用无菌接种循环, 选择一个单一的殖民地, 从琼脂板块。将接种循环放入 LB 介质中, 并简单混合。</li>
		<li>用铝箔盖住锥形烧瓶的顶部, 在37摄氏度的时候用颤抖的一夜孵化。</li>
		<li>用标准 maxiprep 或 midiprep 质粒 dna 纯化试剂盒 (见材料表) 生产转染级质粒 dna10。通过吸光度谱评价 DNA 的质量。 注: DNA 应具有 A260/A280 比 > 1.8 和 A260/A230 比 > 2.0。此时, 建议筛选多个单独的菌落, 以检查插入和标记的有效表达式。</li>
	</ol>
</li>
</ol>2. 细胞培养和转染
注: 对于 BiFC 载体的转染, 重要的是要实现高效, 相对均匀的转染。该载体可能与任何标准转染试剂兼容, 并应相应地优化转染条件。为了进行质谱分析, 我们通常在10厘米的菜肴中培养细胞, 虽然这也可以按比例缩小到较小的盘子或盘子中, 而实验则需要较少的材料。
<ol><li>种子 1.0 x 106 HEK293T 细胞在 10 cm 盘与10毫升 DMEM 媒介, 补充与10% 血清和青霉素或链霉素 (1:100)。</li>
	<li>将每个 BiFC 的2.5 µg 稀释成500µL 的转染缓冲器 (见材料表), 1.5 毫升离心管。</li>
	<li>添加10µL 转染试剂 (见材料表)。</li>
	<li>漩涡混合物为十年代, 然后简要地离心机。室温下孵育10分钟。</li>
	<li>将 DNA 转染的混合物添加到盘子中, 滴状。孵化细胞足够长的时间为 BiFC 融合蛋白相互作用和为金星折叠和荧光成熟, 一般 8-24 h。 注: 金星的峰值激发是 515 nm, 其峰值发射是 528 nm, 虽然这是很容易看到使用标准的荧光显微镜设置, 以可视化 GFP 荧光。</li>
</ol>3. 样品准备
<ol><li>
收割裂解物
	<ol>
<li>在裂解之前收集, 准备细胞裂解缓冲 [50 毫米三盐酸 (pH 7.4), 150 毫米氯化钠, 1 毫米 EDTA, 1% (v/v) 非离子洗涤剂 (见材料表)]。存储在4摄氏度。</li>
		<li>在收割之前, 用蛋白酶抑制剂和磷酸酶抑制剂补充10毫升细胞裂解缓冲液 (见材料表)。在冰上保持细胞溶解缓冲。</li>
		<li>在冰冷的 PBS 中洗两次细胞。吸入 PBS 并加入1毫升的冰寒补充细胞裂解缓冲液。将盘子放在冰上, 确保缓冲区在整个表面积上扩散。</li>
		<li>在冰上孵育大约5分钟, 然后使用一个细胞刮板 (见材料表) 刮的细胞和转移到预冷的1.5 毫升离心管。</li>
		<li>去除细胞碎片通过离心收集的裂解物在 1.8万 x g 5 分钟4摄氏度, 并转移清除上清到新鲜的离心管。 注: 在这一点上, 裂解液可以立即使用, 或储存在-80 摄氏度。此外, 还建议保持整除的粗裂解液, 使转染和亲和纯化的效率得到验证。</li>
	</ol>
</li>
	<li>
亲和纯化 注意: BiCAP 隔离步骤是使用 GFP nanobody 共轭琼脂糖珠 (见材料表) 执行。<ol>
<li>通过洗涤适当的体积 (20 µL 每样品 + 10 µL 过量) 在1毫升 PBS 准备琼脂糖珠。离心的珠子在 300 x g 和删除上清。</li>
		<li>在每个裂解样品中加入20µL。</li>
		<li>以端到端旋转的方法在4摄氏度孵化样品2小时。 注: 在这一点上, 可以为 SDS 页和西方印迹, 或者通过质谱分析来制备样品。</li>
	</ol>
</li>
	<li>
BiCAP 淋洗剂组成的制备。
	<ol>
<li>离心 300 x g的珠子, 在细胞裂解缓冲液中洗涤三次。</li>
		<li>并用重悬在50µL 适当稀释的样品缓冲器 (见材料表), 并加热样品在95°c 2-3 分钟的洗涤珠。 注: 以这种方式制备的样品可在-20 摄氏度内储存几个月。</li>
		<li>对 V1 标签和 V2 标签 (见材料表), 以及任何其他感兴趣的蛋白质, 执行 SDS 页和西部印迹11 。</li>
	</ol>
</li>
	<li>
质谱 BiCAP 淋洗剂组成的制备。 注: 为进行无标签定量 (LFQ) 质谱分析, 建议在至少四份中准备样品, 以确保可靠的统计能力。<ol>
<li>离心的珠子在 300 x g 和洗六次细胞裂解缓冲没有洗涤剂。这是必要的, 以消除多余的蛋白质和洗涤剂, 将干扰质谱分析。立即进行下一步, 或存储在-80 °c 的珠子.
</li>
		<li>胰蛋白酶处理珠在60µL 缓冲 1 [2 M 尿素, 50 毫米三盐酸 (pH 7.5), 5 µg/毫升胰蛋白酶]。允许珠子消化30分钟, 在27°c 在一个 thermomixer 摇晃 800 RPM。</li>
		<li>简要的离心珠, 然后收集上清和转移到离心管 (见材料表)。</li>
		<li>洗涤珠在25µL 缓冲 2 [2 M 尿素, 50 毫米三 HCl (pH 7.5), 1 毫米 dithiothreitol], 为了减少束缚的蛋白质。</li>
		<li>将珠子和上清液池放入一个离心管中。允许消化在室温下过夜。</li>
		<li>烷基化在样品中加入20µL iodoacetamide (5 毫克/毫升的超纯水), 并在黑暗中孵化30分钟。</li>
		<li>用1µL 三氟乙酸酸 (TFA) 处理每个样品, 抑制消化。这一步骤也将酸化的样品准备阶段的小费。</li>
		<li>通过堆叠6层1毫米固相萃取 C18 (C18) 膜盘 (见材料表), 构造出一个200µL 微尖。为每个示例准备一个提示。</li>
		<li>用甲醇湿润舞台提示, 平衡50µL 0.1% (v/v) 三氟乙酸酸 (TFA), 80% (v/v) 乙腈。</li>
		<li>用50µL 0.1% (v/v) TFA 清洗提示。</li>
		<li>将酸化肽加载到舞台尖端, 然后洗脱使用 0.1% (v/v) TFA, 80% (v/v) 乙腈两个步骤。</li>
		<li>使用真空浓缩器蒸发样品。</li>
		<li>如果需要, 将干肽贮存在-80 摄氏度。</li>
	</ol>
</li>
</ol>4. 质谱法。
<ol><li>并用重悬样品在15µL 5% 甲酸, 2% 乙腈 (在超纯水)。</li>
	<li>小心地将6µL 到 LC 板上, 放入 nanoLC 高效液相色谱系统 (见材料表)。</li>
	<li>包装20厘米, 75 µM 内径柱与1.9 µM C18 固定相粒子 (见材料表)。将5µL 肽载入每一列。</li>
	<li>洗脱肽使用乙腈的线性梯度在 250 nL/分钟140分钟以上, 并通过 nanoelectrospray 引入线性陷阱 Quadropole (LTQ) 混合质谱仪与 nanoLC HPLC 系统。</li>
	<li>在140分钟的时间梯度上, 为每个扫描收集10个最丰富的离子的串联 MS 数据。随机的数据收集和交换与 BSA 运行在每个样本之间的顺序, 以尽量减少时间偏差。</li>
</ol>5. 分析
<ol><li>使用 MaxQuant 软件版本1.2.7.4 中的默认设置处理原始 MS 数据, 并在 R 软件环境3中使用修订版本的珀尔修斯统计分析工作流分析 MaxQuant 输出。 注意: 从这一点的统计分析工作流概述在图 2中。简要地, 用 MaxQuant 识别的蛋白质的 LFQ 强度进行了变换和过滤, 然后进行规范化和归一化。二聚体对相互作用的蛋白质通过与金星控制的比较确定, 排除非特异性的背景粘合剂, 与学生的 t 测试和 Benjamini 霍赫贝格校正为多次比较。通过对单个直方图、多重回归和层次聚类的比较, 确定了数据质量。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57109/57109fig2.jpg"> 图 2: 统计分析工作流的概要.统计分析管道流程图, 用于分析使用 MaxQuant 处理的原始质谱数据所识别的蛋白质的 LFQ 强度。绿色框: 过滤, 蓝色盒: 变换/正常化/缩放, 棕盒: 数据质量控制, 黄色框: 半定量排除/比较分析, 灰色盒: 统计分析。这一数字被重新发布 fromCroucher 等3转载的许可, 由科学促进会。<a href="//cloudflare.jove.com/files/ftp_upload/57109/57109fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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作者没有什么可透露的

BiCAP 是一种有效的分离特定蛋白脂肪酸的方法, 而不包括单个成分及其相互竞争的结合伙伴3。BiCAP 是基于一种称为 BiFC6的荧光蛋白互补试验的适应性。现有的方法, 包括 BiFC 和近距离结扎法, 已广泛用于可视化和量化活细胞中的蛋白质相互作用7, 但没有提供一种有效的方法来分离和表征脂肪酸形成的这些相互作用。
基于蛋?...

蛋白质复合组装是维持许多信号通路1、2的时空特异性的关键过程。虽然这一管制作用的关键性质得到广泛承认, 但缺乏用于审查这些复合体的实验技术。大多数 interactomics 研究的重点是与个体蛋白质的相互作用, 或单个复杂成分的顺序浓缩。在这里, 我们提出了一种分离特定蛋白质二聚体的技术, 而不包括单个基团的成分蛋白, 以及形成了与竞争的捆绑合作伙伴3。我们称之为这种技术分子互补亲和纯化 (BiCAP), 因为它是一个以前现有的蛋白质片段互补检测, 分子荧光互补 (BiFC) 的组合, 与新的使用构象特异重组 nanobody 对 GFP 及其衍生物 (见材料表).
一个典型的蛋白质片段互补试验依赖于 "诱饵" 和 "猎物" 蛋白的表达, 融合成分裂的记者, 如荧光素酶4, β-乳糖酶5, 或绿色荧光蛋白 (GFP)6 (图 1A)。通过诱饵和猎物蛋白的相互作用, 鼓励分裂的记者领域再折叠餐巾成一个功能结构, 允许诱饵和猎物蛋白的相互作用被可视化或量化。BiCAP 是根据这种技术的版本改编的, 利用了 GFP 变种金星的片段。荧光蛋白互补检测是一种常见的方法, 以可视化的蛋白质-蛋白质相互作用的活细胞, 但直到现在已被限制在这一功能7。BiCAP 代表了这方面的一个重大进展, 因为这项技术不仅允许可视化, 而且还对由此产生的蛋白质-蛋白质相互作用的隔离和审问。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57109/57109fig1.jpg"> 图 1: BiCAP 技术背后的结构主体.(a) 概述分子荧光互补原理的示意图, 显示 "诱饵" 和 "猎物" 蛋白, 标签为 n-端 V1 或 V2 的全长金星蛋白片段。(B) 对 GFP nanobody (绿色) 与重组金星之间的交互界面 (青色) 进行结构分析, 显示 V1 (灰色) 和 V2 (橙色) 片段 (PDB 加入 3OGO) 的位置。这一数字被重新发布 fromCroucher 等3转载的许可, 由科学促进会。<a href="//cloudflare.jove.com/files/ftp_upload/57109/57109fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
BiCAP 技术利用金星的两个非荧光片段 (命名为 V1 和 V2), 它与低亲和力关联, 除非它们的融合伙伴之间发生交互作用。在此实例中, 两个拆分域再折叠餐巾到荧光的功能β桶结构 (图 1B)6。BiCAP 的关键创新来自于对重组 gfp nanobody 的介绍, 它能识别出 gfp (和金星等变种) 的β桶上的三维表位, 这只存在于正确重组和折叠的荧光 (图 1B)8。关键的是, GFP nanobody 不绑定到单独的金星碎片之一。这促进了蛋白质脂肪酸的分离, 只有在这两种蛋白质形成了自己的意志复杂, 导致更具代表性的结果比从方法, 利用化学诱导, 强迫互动9。
BiCAP 是一种功能强大的技术, 专门专注于多蛋白复合物, 这可能与一些下游应用结合, 以提高我们对这些复合体在信号传导中所起的作用的理解的粒度。.它还包括允许在原位进行蛋白质相互作用可视化的重要特征。迄今为止, BiCAP 已被证明是一种有效的方法来分析受体酪氨酸激酶 (RTK) 脂肪酸3的 interactome, 但这种方法的适应性意味着它可以在几乎任何蛋白质相互作用的情况下采取。

<table><tbody><tr><td>LR Clonase II Plus enzyme</td><td>Thermo Fisher Scientific</td><td>12538120</td><td>Recombinase enzyme required for Gateway cloning (Step 1) into pDEST BiFC destination vectors</td></tr><tr><td>Proteinase K, recombinant, PCR grade</td><td>Thermo Fisher Scientific</td><td>EO0491</td><td></td></tr><tr><td>14 mL round-bottomed polypropylene tube</td><td>Corning</td><td>352059</td><td></td></tr><tr><td>Ampicillin</td><td>Roche Diagnostics Australia</td><td>10835242001</td><td>Stock solution prepared at 100 &amp;mu;g/mL in distilled water.</td></tr><tr><td>Miniprep kit</td><td>Promega Corporation</td><td>A1330</td><td></td></tr><tr><td>Maxiprep kit</td><td>Life Technologies Australia</td><td>K2100-07</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995-073</td><td></td></tr><tr><td>FBS</td><td>Life Technologies Australia</td><td>10099-141</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies Australia</td><td>15070-063</td><td></td></tr><tr><td>jetPRIME transfection buffer</td><td>Polyplus</td><td>114-15</td><td></td></tr><tr><td>jetPRIME transfection reagent</td><td>Polyplus</td><td>114-15</td><td></td></tr><tr><td>PhosSTOP (Phosphatase inhibitor)</td><td>Sigma-Aldrich</td><td>4906837001</td><td></td></tr><tr><td>cOmplete, Mini, EDTA-free Protease inhibitor cocktail</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Cell Scraper</td><td>Sarstedt</td><td>83.1832</td><td></td></tr><tr><td>GFP-Trap_A</td><td>Chromotek Gmbh</td><td>gta-100</td><td>GFP nanobody coupled to agarose beads</td></tr><tr><td>N-terminal GFP monoclonal antibody&amp;nbsp;</td><td>Covance</td><td>MMS-118P</td><td>Will detect the V1 tag within the BiFC vectors</td></tr><tr><td>C-terminal GFP monoclonal antibody</td><td>Roche</td><td>11814460001</td><td>Will detect the V2 tag within the BiFC vectors</td></tr><tr><td>Sample buffer</td><td>Invitrogen</td><td>NP0008</td><td>Supplemented with 1 mL &amp;beta;-mercaptoethanol.</td></tr><tr><td>Sequencing grade modified trypsin</td><td>Promega Corporation</td><td>V5117h</td><td></td></tr><tr><td>LoBind microcentrifuge tubes</td><td>Point of Care Diagnostics</td><td>0030 108 116</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I1149-5G</td><td>Prepared at 5 mg/mL in ultrapure water</td></tr><tr><td>Trifluoroacetic Acid - Sequanal Grade</td><td>Thermo Fisher</td><td>10628494</td><td></td></tr><tr><td>3M Empore solid phase extraction C18 disks (octadecyl) - 4.7 cm</td><td>Thermo Fisher</td><td>14-386-2</td><td>To prepare stage tips, cut 1 mm disks using an appropriately sized hole punch. Alternatively, pre-prepared stage tips can also be purchased, see below.</td></tr><tr><td>C18 Stage Tips, 10 &amp;micro;L bed</td><td>Thermo Fisher</td><td>87782</td><td></td></tr><tr><td>Formic acid OPTIMA for LC/MS grade 50mL</td><td>Thermo Fisher</td><td>FSBA117-50</td><td></td></tr><tr><td>1.9 &amp;mu;m C18 ReproSil particles</td><td>Dr. Maisch GmbH</td><td>r119.aq.</td><td>Stationary phase particles</td></tr><tr><td>Acetonitrile OPTIMA LC/MS grade&amp;nbsp;</td><td>Thermo Fisher</td><td>FSBA955-4</td><td></td></tr><tr><td>Easy-nLC HPLC&amp;nbsp;</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>LTQ Orbitrap Velos Pro</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Non-ionic detergent (100%)</td></tr><tr><td>DH5&amp;alpha; cells</td><td>Thermo Fisher</td><td></td><td>Heat-shock-competent cells</td></tr></tbody></table>

dissecting multi-protein signaling complexes by bimolecular complementation affinity purification (bicap)

蛋白质复合体的组装是许多细胞信号通路调控的中心机制。生物医学研究的一个主要重点是破译这些动态蛋白复合物如何将信号从多个来源整合起来, 以指导特定的生物反应, 以及如何在许多疾病的环境中解除管制。尽管这一关键的生化机制的重要性, 缺乏的实验技术, 可以促进具体和敏感的反褶积这些多分子信号复合物。
在这里, 这一缺陷的解决方法是通过蛋白质互补试验与构象特异 nanobody 的结合, 我们称之为分子互补亲和纯化 (BiCAP)。这种新的技术促进了特定的隔离和下游蛋白质组特征的任何一对相互作用的蛋白质, 排除不复杂的个体蛋白和复合物形成与竞争的捆绑合作伙伴。
BiCAP 技术适用于广泛的下游实验测试, 这种技术提供的高度特异性使得对蛋白质复杂装配的力学的更细致的研究比目前可能使用标准亲和纯化技术。

在使用重组克隆产生的 V1 和 V2 标记基因感兴趣的 BiFC pDEST 质粒, 共转染两个质粒含有相互作用对蛋白质将导致产生金星荧光信号后,大约8-24 小时。在没有阳性信号的情况下, 由于细胞系的选择、转染效率低或 BiFC 标记的方向不理想, 蛋白质相互作用可能不会发生。下面将讨论每个方案的疑难解答。
我们以前观察到, 在 HEK-293T 细...

Watch this Scientific Journal Video about Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP at JoVE.com

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

本手稿描述了分子互补亲和纯化 (BiCAP) 的协议。这种新方法促进了任何两种相互作用的蛋白质的特定隔离和下游蛋白质组特征, 同时排除了不复杂的个体蛋白质以及与竞争的结合伙伴形成的复合体。

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

dissecting-multi-protein-signaling-complexes-bimolecular

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

7.4K 次观看.  Garvan Institute of Medical Research. 本手稿描述了分子互补亲和纯化 (BiCAP) 的协议。这种新方法促进了任何两种相互作用的蛋白质的特定隔离和下游蛋白质组特征, 同时排除了不复杂的个体蛋白质以及与竞争的结合伙伴形成的复合体。

视频: Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

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

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

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 ...

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 ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

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 ...

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

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

双分子荧光互补

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

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 ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

完整蛋白质复合物的化学交联和质谱分析研究多亚基蛋白质组合体的体系结构

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

用于研究蛋白质-蛋白质相互作用的串联亲和纯化检测

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...