这项工作得到了麦迪逊和莱拉自学奖学金 (AJM)、nih 5T32GM008359、KUMC 生物医学研究培训计划、nih R01AI090085 (MTF)、KU 架桥基金和分子互动技术中心 (社区商业) 赠款的支持 (CT 和 MTF)。作者想感谢芭芭拉 Fegley 在 KUMC 电子显微镜的核心协助与 TEM 图像采集。

1. Biolayer 干涉测量 (BLI) 生物传感器表面上定义的大分子络合物的组装
<ol><li>
PDEA 修饰胺反应生物传感器表面 prepore 炭疽毒素复合物的组装
	<ol>
<li>水合胺反应性第二代 (AR2G) BLI 生物传感器提示在250µL 水中十分钟。</li>
		<li>程序步骤时间, 列在表 1中, 关于控制 BLI 单元的软件 (见材料表)。</li>
		<li>通过将生物传感器尖端浸泡在三十年代的250µL 水中, 开始 BLI 运行, 以测量生物传感器厚度和密度的初始基线。</li>
		<li>通过将尖端浸入250µL 50 毫米 NHS (n-琥珀) 和200毫米 (1-乙基-3-(3-dimethylaminopropyl) carbodiimide), 激活生物传感器7分钟。</li>
		<li>将激活的生物传感器浸入50毫米 PDEA (2-(2-pyridinyldithio) 乙胺), 溶解在0.1 米硼酸盐缓冲液 (pH 8.5) 中, 5 分钟产生活性的硫醇反应表面。</li>
		<li>将活化的硫醇反应生物传感器浸入250µL 溶液中, 含 100 nM E126C, 10 毫米醋酸钠 pH 5.0, 100 毫米氯化钠, 缓冲5分钟。</li>
		<li>将 LFN尖浸入50毫米 l-半胱氨酸, 1 米氯化钠, 0.1 米醋酸钠, pH 5.0, 4 分钟, 以淬火任何剩余的无活性的硫醇反应基团。</li>
		<li>将淬火的 LFN尖浸入50毫米, 50 毫米氯化钠, 2 分钟以建立缓冲基线。</li>
		<li>将淬火的 ifn尖端浸入0.5 µM 保护性抗原 prepore (PAprepore), 50 毫米三, 50mM 氯化钠5分钟, 以创建 ifn-PAprepore复合物。</li>
		<li>一旦 paprepore相关联, 从 paprepore溶液中取出尖端, 将尖端浸入50毫米三, 50 毫米氯化钠30秒, 以冲洗掉任何非特定束缚的 PAprepore。</li>
		<li>将 LFprepore复合物浸入0.5 µM CMG2 受体 (无跨膜区), 50 毫米三, 50 毫米氯化钠, 5 分钟.</li>
		<li>将 LFn-PAprepore-CMG2 络合物浸入50毫米, 50 毫米氯化钠30秒, 冲洗掉任何未绑定的 CMG2, 形成预细胞膜复合体。</li>
		<li>对于 EM 分析, 从生物传感器尖端释放 LFn-PAprepore-CMG2 络合物, 将尖端浸入5µL 50 毫米, 50 毫米三, 50 毫米氯化钠, 在 PCR 管内。</li>
		<li>对复合体中多肽的串联 MS 分析, 将生物传感器浸入5µL 50 毫米, 6 米图法分别 (无角蛋白), 在 PCR 管内的25毫米碳酸氢铵 pH 值 8.0, 从生物传感器尖端释放 LFprepore-CMG2 络合物。.这是在不同的生物传感器上执行的, 而不是用于 EM 分析。</li>
	</ol>
</li>
	<li>
PDEA 改性胺反应生物传感器表面上孔型炭疽毒素复合物的组装
	<ol>
<li>要查看 pH 值转换后的复杂情况, 请执行上一节中的步骤 1.1. 1-1. 1.12, 以生成 ifn-PAprepore-CMG2 复合体。</li>
		<li>将生物传感器尖端浸入10毫米醋酸盐 pH 值 5.0 5 分钟, 启动 paprepore向 pa孔过渡的过渡。这个转折由增加的振幅 (大约0.2 毫微米) 指示, 然后一个更大的振幅减退被假设是坚固或完全受体离解由于束缚亲和性的减少。</li>
		<li>浸没在50 毫米三, 50 毫米氯化钠, pH 8.0, 30 秒冲洗酸性缓冲液中的孔尖。</li>
		<li>对于 EM 分析样本, 将生物传感器尖端浸入一个包含1.25 毫米胶束 (2.5 毫米 MSP1D1、25毫米 Na-胆酸、162.5 毫米 POPC) 的溶液中, 以防止在二硫化物释放后溶液中聚集。</li>
		<li>对于 EM 分析, 从生物传感器尖端释放 LFn-PA孔隙胶束复合物, 将尖端浸入5µL 50 毫米, 50 毫米三, 50 毫米氯化钠内的 PCR 管内。</li>
		<li>对复合体中多肽的串联 MS 分析, 通过将生物传感器浸入5µL 50 毫米, 6 米图法分别 (无角蛋白)、25毫米碳酸铵 pH 值8.0 内的 PCR 方法, 从生物传感器尖端释放 LFn-PA可怜复合物 (无胶束)。管。这是在不同的生物传感器上执行的, 而不是用于 EM 分析。</li>
	</ol>
</li>
</ol>2. 用负染色电子显微镜对 BLI 生物传感器释放的大分子组件进行可视化和验证
<ol><li>辉光放电碳涂层铜300网格。典型的辉光放电设置是0.38 毫巴稳定的气压, 负 15 mAmps, 20 秒, 然后与空气排气。</li>
	<li>在一对干净的镊子之间的安全网格。</li>
	<li>吸管4µL 在 PCR 管中释放的复合样品进入网格, 并允许吸附60s。</li>
	<li>用过滤纸楔将剩余液体吸走。</li>
	<li>通过吹打5µL 0.75% 0.02 微米的甲酸酯过滤并在5秒后吸走多余的污渍, 使网格着色。允许网格在室温下干燥。</li>
	<li>使用透射电子显微镜查看染色样品网格。</li>
</ol>3. 用质谱法鉴定完整的细胞膜炭疽毒素复合体 (lfprepore-CMG2) 和变性复合体 (lf 无 CMG2 ).
<ol><li>稀释释放的样品到20µL 和孵育1小时。</li>
	<li>添加2µL 55 毫米 iodoacetamide, 25 毫米碳酸氢铵, pH 8.0, 并在室温下孵育1小时 (覆盖铝箔)。</li>
	<li>稀释样品与100µL 25 毫米碳酸氢铵, pH 8.0, 减少盐酸胍浓度在 1 m 以下。</li>
	<li>添加5µL 的顺序级改性胰蛋白酶在 20 ng/µL 和孵化在37°c 过夜。</li>
	<li>加入冰乙酸到最后浓度为 5%, 以减少 pH 值 < 3, 然后减少体积到10µL 在真空浓缩器。</li>
	<li>将肽溶液转移到 nLC 1200 uHPLC autosampler 的样品板上。</li>
	<li>负载5µL 肽溶液到 uHPLC 反相柱上安装在 MS 的电离阶段。</li>
	<li>洗涤柱与15µL 0.1% 甲酸在最大速率 5 µ升/分和/或最大压力 800 psi。</li>
	<li>洗脱肽从反相 C18 柱的流速为 350 nL/分钟在90分钟内使用的线性梯度5% 到40% 的溶剂 b 在 a + B (溶剂 a: 0.1% 甲酸; 溶剂 b: 95% 乙腈与0.1% 甲酸)。</li>
	<li>用串联 MS 对洗脱肽进行在线分析。<ol>
<li>将电离源设置为2500伏特和离子转移温度为250摄氏度。</li>
		<li>在自动控制下运行质谱仪连续进行一毫秒扫描, 在3秒内尽可能多串联 MSMS, 使用 CID 和规范化的碰撞能量35。</li>
	</ol>
</li>
	<li>使用标准方法识别肽和蛋白质成分。对这项工作进行了两组肽和蛋白质分析。</li>
</ol>

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此时没有披露。

这一演示说明了如何使用 biolayer 干涉仪, 用 EM 可视化的方法来方便地监测大分子复杂地层, 并在短时间内对 microvolumes 进行了验证。结构组装和观测到的复合体遵循生物相关的预测, 进一步验证了这种组合方法。如结果部分所述, 组装成功的关键因素需要合理地设计半胱氨酸突变, 以确保复杂的蛋白质-蛋白质界面能正确地定向远离生物传感器表面。
以前的系统已经使用 BLI 和 MS 技术来评估两个和三组分系统的蛋白质结合以及表达蛋白的受体结合的完整性, 但在这两种情况下, 都没有开发出利用串联 EM 和 ms 的方法。接近8,9。唯一的其他干涉系统, 结合 MS 分析, 以帮助表征相互作用是双重极化干涉测量10。不幸的是, 这个系统已不再适用于一般用途。正如导言中所提到的, 已经完成了一些研究, 其中样本是在 spr 型生物传感器表面形成的, 并从 MS 分析中去除。这些例子中没有一个结果是用 EM 可视化的复合体。
这种顺序方法的局限性可以是很多的, 但是可解的。对于装配初期的固定化和基础步骤, 缺乏对结构相互作用表面的知识肯定会阻碍与监测初始装配阶段有关的进展。结构信息的缺乏可以通过设计一个工程基础结构来解决, 其中附着化学 (如硫醚的巯基基团/他标记的定位) 可以移动到核心中的各个区域。装配系统。在炭疽毒素复合体的情况下, 幸运的是, LFN的结构与 PAprepore是可用的11。将工程半胱氨酸的合理位置定位到远离 PAprepore结合面的区域。与半胱氨酸连锁化学, 最好的是没有其他反应半胱氨酸在蛋白质表面存在。
有各种附着化学物, 可用于在蛋白质表面上设计特定的附着部位。最受欢迎的特定附件之一是将生物素基团放置在蛋白质表面12的特定位置上。不幸的是, 生物素结合到链亲和素或亲和涂层表面是相当紧密的。反向绑定交互并不简单。在 N 或 C 终点使用工程化的他的标签, 随后容易附着到镍 NTA 表面上, 是一种更为普遍的亲和固定化应用。当然, 工程装配附件站点与他标记的系统的一个警告是要求核心装配蛋白的 N 和 C 终点保持暴露和分离, 以便附件是容易的。与所有的装配过程一样, 核心组装蛋白的相互作用界面必须在复杂的组件进展时保持可用。
也许使用生物传感器表面化学的最常见的问题是非特定的结合。链亲和素提示通常是重要的非特定绑定效果的来源。二硫化物链接生物素可以用来释放非常具体的复合物留下的减少 S-生物素联系紧密绑定到固定化链亲和素生物传感器13。还有其他可逆化学制剂, 如 iminoboronates 和在较小程度上 ketoamide14。这个领域目前不发达, 但有很大的兴趣进一步发展可逆的共价键, 以避免非靶向药物毒性效应, 通常伴随使用共价靶向药物开发。
使用 EM 来可视化复合物的限制是解释, 特别是在组装复合体的结构最初不知道的情况下。用单克隆抗体作为特定的动力学和结构标记, 可以识别未定义的组装大分子复合物中元件的空间位置。例如, 一旦形成一个复杂的, 单克隆抗体可以添加绑定到特定的组件。此方法经常用于 EM 以识别大型组件15中的特定组件。另一个限制与复合体的大小有关, 尽管有实例, 即定义的对称程序集小到 70 kDa (GroES heptamer) 很容易用负污点 EM 解决。由 EM 分析的组装配合物通常在直径或以上的100Å的大小范围内。然而最近, 20 kDa 的蛋白质已经得到了解决, 并且在使用高级染色方法16时获得了低分辨率结构。
对于 ms 分析, 目前 ms 仪器的灵敏度提高到 femtomolar (attomoles) 水平, 在某些情况下可以提高 BLI 检测的灵敏度。很有可能的是, 蛋白质信号, 显示一个极小的, 但可重复上升的振幅将导致识别的蛋白质的问题。此外, 探讨蛋白质与蛋白质的相互作用, 其中一个伙伴附着在生物传感器和其他在细胞环境中, 将有效地导致净化和随后更容易检测新形成的复杂。目前高度敏感的 MS 系统可以观察到的一个局限性是, 感兴趣的蛋白质可能不在数据库中, 但这种观察是罕见的 (例如,稀有物种的蛋白质组)。如果有兴趣的蛋白质序列已知, 这个问题很容易解决, 包括蛋白质 (s) 氨基酸序列的背景蛋白数据库 (如本工作在协议部分)。这种方法的另一个潜在的局限性是由蛋白质对 trypsinolysis 的抵抗力造成的。胰蛋白酶消化通常是自下而上蛋白质鉴定的默认方法。然而, 蛋白质可能对胰蛋白酶有抗药性, 如果他们缺乏 Arg 和赖氨酸残留物或获得这些残留物受到折叠结构的限制。这些限制分别是通过使用替代或组合蛋白酶或包括展开试剂 (如1.1.14 和1.2.6 中所示) 在酶消化之前。
这种方法的可能扩展包括允许用户跟踪和识别来自粗细胞裂解物的细胞组装复合物。结果表明, 在浓缩细胞萃取物中组装元件的测试很容易使用 biolayer 干涉测量程序进行。与更常用的基于微流控的方法相比, 容易堵塞和对聚集敏感, BLI 方法可用于直接将 biolayer 传感器提示浸入粗萃取物中, 从而有可能直接装配特定的络合物。从这些浓缩的不纯样品。一旦组装, 使用特定的抗体探针作为 BLI 系统的后续行动是完全可行的, 以进一步识别, 甚至定量可疑成分的细胞提取物, 已识别使用 microvolume MS 方法。再次, 这里的关键是使用定义的, 适当地定向核心蛋白作为特定的亲和探针。
动力学与 BLI 的 prepore 孔过渡过程的能力, 将非常有用的发现潜在的 "抗毒素" 小分子抑制剂的蛋白质转变, 特别是功能在后期细胞膜, 低 pH 值 (5.0)条件。这种 pH 诱导的 prepore 孔转变是抑制在存在的折叠稳定剂 (渗透调节物质), 如甘油或蔗糖, 从而为开发特定的目标折叠稳定剂, 以防止 PA孔形成的强大支持。这种特殊的方法避免和取代了基于粗糙聚合的化验, pH 值下降导致蛋白质沉淀。后一种方法, 虽然很好的主要预检方法, 往往导致假阳性结果, 其中特定化合物抑制聚集, 而不是实际的分子转变。
下游观测的结构和鉴定的个别组装部件在小 microvolume 样品也可以用于验证潜在的铅化合物。在特定的程序集稳定或不稳定是目标结果的情况下, 可以应用此方法。这种动力学/结构/识别并行方法有助于直接确认可疑铅复合效应剂的有效性, 并作为一个合理的二次筛选步骤或中等吞吐量方法。
低温 EM 是研究不同组装状态下大分子络合物的原子细节的一种有用的技术。在制备低温 em 样品之前, 首先要验证制备中是否含有带有负染色的纯同质复合物。本文介绍的工作表明 BLI 生物传感器表面的蛋白质复合物的组装, 这些配合物的释放为 EM 可视化, 并使用 MS 识别这些成分。这种控制装配和释放的特殊方法可以用于生成非常具体的协议, 以增强对负染色 EM 的均质序贯样品准备, 这是必须在推进到冷冻 EM为了获得低分辨率的3D 结构, 只有30-50 粒子的复杂将需要执行一个圆锥倾斜系列 (70 不同的2D 图像视图每个粒子), 只要有方向多样性 (多个不同的看法)。
在提高 MS 方法方面, 灵敏度和样品量减少的进展继续改善。纳米流与超高压液相色谱法一起开发了质谱仪, 具有快速的工作周期, 提高了灵敏度和分辨力。最近介绍的 orbitrap 质谱仪, 特别是最新版本 (orbitrap 融合 Lumos, 及其预期的后继者 orbitrap 融合 Lumos 1M) 以及搜索算法极大地促进了这一过程。
目前的方法是使用无标签的 BLI 方法监测炭疽毒素成分的动态组装和拆卸, 并分别使用 EM 和 MS 对这些组分的结构和特性进行评估。采用简单的单通道 BLI 系统, 加上常规的负染色 EM 分析和初等 MS 技术, 足以表征装配过程。

识别和理解体内蛋白质复合体的特异性对生物化学研究人员是极其感兴趣的。大型异构蛋白组件是规范而非例外。这个概念是支持的光谱监测体内组装, 隔离配合物使用温和的细胞中断技术, 评估产品从亲和性的纯化方法, 并可视化他们使用高分辨率低温电子显微镜 (冷冻 EM)。为了了解细胞内装配特异性的控制, 研究人员必须例行地隔离、识别和最终描述这些动态组装/拆卸结构。最常用的分子工具, 以识别这些组件的组成部分往往需要基于抗体的免疫沉淀, 这依赖于维持复杂的稳定性在细胞中断。最近开发了各种耦合分析技术, 利用微流控方法, 如表面等离子体共振 (SPR) 从细胞样品中捕获复合物。从 SPR 表面去除后, 通过基质辅助激光解吸/电离飞行时间 (MALDI)1、2对这些样品进行了分析。使用更简单的协议推进这种方法将使研究人员能够想象和验证在细胞环境中发生的预测复合物。由于 SPR 是一个基于微流体的系统, 所以问题往往是由聚集形成引起的。规避这个问题需要样品稀释, 这反过来, 可以降低浓度的生物复合物的完整性。
无标签技术的一个相对最近的进展是 biolayer 干涉测量 (BLI) 系统的发展3。这些特定的光反射系统复制, 或最佳模拟, SPR 结合和动能结果在成本的3,4的一部分, 特别是如果使用单通道单位。BLI 测量参照层 (控制) 和 biolayer 表面之间反射光干涉模式的变化 (实验)。所产生的相位变化是实时测量的, 作为一个动力学和定量读数5。生物传感器表面, 含有特定的固定化化学物质, 在溶液之间物理转移, 而不是通过微流控方法在 SPR 中的缓冲变化, 通过波长相位偏转测量。通过搅拌溶液来防止传质效应。与 SPR 不同, 这些系统在评估来自粗生物样品的复合物方面非常有用。在 BLI 实验中测量的物理参数主要取决于生物传感器表面的质量或厚度的变化, 这是由蛋白质复杂的组装或拆卸造成的。
这些光纤生物传感器易于使用, 相对便宜。BLI 的一个新兴的有用方面是从表面上去除新组装的蛋白质复合物。该方法的最新应用使我们的实验室能够观察到炭疽毒素保护抗原 (pa) 组分的大尺度 pH 诱导蛋白结构重排的实时动力学, prepore (paprepore) 转化为其孔 (PA孔) 形式。利用电子显微镜 (EM)6对生物传感器尖端的过渡进行了验证。从生物传感器表面去除复合物, 避免了在使用微流体系统时从芯片表面释放复合物时经常遇到的体积稀释效应。
在目前的工作中, 炭疽毒素复合物在生物传感器表面组装和拆卸, 然后释放成微升体积。使用 EM 和质谱 (MS) 正交验证了所产生的复杂成分。

<table><tbody><tr><td>(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)) (EDC)</td><td>Thermo Scientific</td><td>22980</td><td></td></tr><tr><td>(2-(2-pyridinyldithio)ethanamine) (PDEA)</td><td>GE Lifesciences</td><td>BR100058</td><td></td></tr><tr><td>0.5 mL black microtubes</td><td>Sigma-Aldrich</td><td>Z688304-500EA</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td><td>Avanti</td><td>850457C-25mg</td><td></td></tr><tr><td>Acclaim PepMap 50 &amp;mu;m x 150mm, C18, 2 &amp;mu;m, 100 &amp;Aring;</td><td>ThermoFisher Scientific</td><td>164561</td><td></td></tr><tr><td>Acetic acid glacial</td><td>Fisher</td><td>A38SL-212</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>A955-4</td><td></td></tr><tr><td>Amine reactive 2nd generation (AR2G) tips</td><td>Forte Bio</td><td>18-5092</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Fisher</td><td>A643-500</td><td></td></tr><tr><td>Anotop 10 syringe filter, 0.02um</td><td>GE Lifesciences</td><td>6809-1002</td><td></td></tr><tr><td>BLItz System</td><td>Pall Forte Bio</td><td>45-5000</td><td></td></tr><tr><td>Boric acid</td><td>Fisher Scientific</td><td>10043-35-3</td><td></td></tr><tr><td>Capillary Morphogenic Gene-2</td><td></td><td></td><td>Protein produced and purified in-house</td></tr><tr><td>Carbon coated Cu 300 grid</td><td>Electron Microscopy Sciences</td><td>CF300-CU-50</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>GoldBio</td><td>DTT25</td><td></td></tr><tr><td>Formic acids</td><td>JT Baker</td><td>0129-01</td><td></td></tr><tr><td>Guanidine hydrochloride (GuHCl)</td><td>Sigma-Aldrich</td><td>G4505-100G</td><td></td></tr><tr><td>Iodoacetamide</td><td>MP-BioMedicals,LLC</td><td>100-351</td><td></td></tr><tr><td>JEM-1400 Transmission Electron Microscope</td><td>JEOL</td><td></td><td></td></tr><tr><td>L-cystiene hydrochloride hydrate</td><td>Sigma-Aldrich</td><td>C121800-5G</td><td></td></tr><tr><td>Lethal Factor N-terminal domain</td><td></td><td></td><td>Protein produced and purified in-house</td></tr><tr><td>MS software version 2.2</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>N-hydroxysuccinimide (NHS)</td><td>Sigma-Aldrich</td><td>090M14531V</td><td></td></tr><tr><td>Orbitrap Fusion Lumos</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>PCR tubes</td><td>ThermoFisher Scientific</td><td>AB-0620</td><td></td></tr><tr><td>Pelco easiGlow glow discharge cleaning system</td><td>Ted Pella, Inc</td><td>91000</td><td></td></tr><tr><td>Protective Antigen prepore</td><td></td><td></td><td>Protein produced and purified in-house</td></tr><tr><td>Qualitative filter paper circles</td><td>Fisher Scientific</td><td>09-795C 5</td><td></td></tr><tr><td>Sequencing grade modified trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Sequest HT search engine</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>Fisher Scientific</td><td>BP334-500</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S271-10</td><td></td></tr><tr><td>Sodium cholate</td><td>Sigma-Aldrich</td><td>C1254-100G</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-10</td><td></td></tr><tr><td>Uranyl formate (UF)</td><td>Electron Microscopy Sciences</td><td>22450</td><td></td></tr><tr><td>Xcalibur</td><td>ThermoFisher Scientific</td><td>OPTON-30487</td><td></td></tr></tbody></table>

analyzing dynamic protein complexes assembled on and released from biolayer interferometry biosensor using mass spectrometry and electron microscopy

在体内,蛋白质通常是大型大分子复合体的一部分, 结合特异性和动力学最终决定功能输出。在这项工作中, 预细胞膜炭疽毒素被组装并转化为细胞膜复合体。首先, 半胱氨酸突变体致死因子 (LFn) 的 N 端域附着在一个 biolayer 干涉仪 (BLI) 生物传感器上, 通过二硫化物耦合在一个最佳方向, 允许保护性抗原 (PA) prepore 绑定 (Kd 1 nM)。最优化的 LFn-PAprepore复合物然后结合到可溶性毛细管形态 gene-2 (CMG2) 细胞表面受体 (Kd 170 pM), 导致一个代表性的炭疽前细胞膜复合物, 稳定在 pH 7.5。这个装配的复合体然后遭受酸化 (pH 5.0) 代表晚 endosome 环境转移 PAprepore入膜插入的孔状态。这种 PA孔隙状态导致了 CMG2 受体与 LFn-PA孔隙之间的束缚, 以及 CMG2 从过渡孔隙中的大量离解。dithiothreitol 可以很容易地逆转 LFN与生物传感器表面的硫的附着。BLI 生物传感器表面的还原释放出 lfn-paprepore-CMG2 三元复合物或酸转化为微升体积的n-pa孔隙络合物。释放的复合物然后可视化和识别使用电子显微镜和质谱。这些实验演示了如何用无标签的 BLI 方法监测特定蛋白质复合物的动力学组装/拆卸, 并通过电子显微和质量评价这些 BLI 组装配合物的结构和特性。分光光度法, 分别使用易于复制的顺序程序。

对大型大分子络合物的组装进行监测和验证的能力是了解大型生物分子组件的特异性和功能的关键步骤。本文提出的方法的结果表明, 使用 biolayer 干涉法组装大型蛋白质复合体 (> 150 kDa 质量) 是很容易的, 同时监测装配的动力学和振幅。生物传感器表面独特的紧凑性使得装配分析可以通过将组装的复合物释放到小 microvolumes 来扩展。这些 microvolumes 可用于可视化复合体的初始物理结构, 并使用 MS 验证复杂元件的标识。图 1显示了整个过程的示意图。
在生物传感器表面上成功组装和验证大分子络合物的一个关键因素是初始种子蛋白的正确定位。这保证了蛋白质-蛋白质相互作用的站点是可利用的, 不 sterically 阻拦, 并且优选地被安置离生物传感器表面。如图 2所示, 炭疽毒素复合体的正确定位是通过使用特定工程的致命因子 (lf n) 的 n 终端片段来实现的, 所以如果prepore绑定站点始终位于在生物传感器共价附件站点6,7对面。随后建立的复合体结合 pa 到 LFN BLI 生物传感器后, 可溶 CMG2 结合到 PA 最终产生一个易位主管炭疽毒素复合物。
BLI sensogram 跟踪是一个实时读取的振幅变化, 由于特定的添加炭疽毒素成分, 因为它们被添加到生物传感器。图 3显示了一个具有代表性的跟踪, 该模型在该过程中被预测为形成的复合体。第一个上升是, 如果N加载到尖端。在淬火和基线后, PAprepore然后结合到 lfn , 然后加入可溶性 CMG2 受体, 导致细胞膜复合体的 lfpreporeCMG2.为了在晚期 endosome 环境中取得进展, 整个复合体受到低 ph 脉 (ph 值 5.0) 的削弱, 从而减弱受体结合, 使 prepore 过渡到其扩展的膜插入孔构象。6 Sensogram 的酸化步骤的痕迹如图 4所示。振幅的初始增加或 "穗" 很可能是在 CMG2 受体结合减少之前必须发生的孔隙扩展6 。更大的振幅下降是最有可能的实质性或完全受体离解由于束缚亲和性减弱。本实验室先前的工作表明, 与 CMG2-PAprepore相互作用6相比, CMG2 对完全扩展的 PA孔隙的束缚是微不足道的。此外, sensogram 的动力学轨迹, 观察在所有 sensograms 的prepore-CMG2 过渡到 lfn-pa孔与 pH 值下降, 是可重复的多次运行。
在酸化之前和之后, 生物传感器附着的复合物很容易被释放出来, 由负染色 EM 和识别的 MS (图 5)。来自 EM 结果的代表性复合体如图 6所示。预细胞膜样品网格, 显示密度与完整的三元复合物相一致, 由 LFprepore-CMG2 组成。酸化后的复杂网格, 显示 PA 过渡到孔和可溶性的胶束夹杂, 没有明显的 CMG2 密度。
用 MS 验证了酸化前后复合物的特性。一个数据库, 其中的 PA 序列, P13423;如果, P15917;和 CMG2, P58335 被纳入了从 NCBInr 存储库派生的鼠标蛋白质数据库的背景。只有感兴趣的蛋白质从这第一个数据库查寻获得, 以以下氨基酸覆盖面为三元和二进制复合体: 54% 和22% 为 PA, 36% 和6% 为 LF 和43% 为 CMG2, 分别 (CMG2 未被发现在二进制复杂)。为了最大限度地提高蛋白质氨基酸的覆盖率, 第二肽/蛋白质的鉴定是使用一个蛋白质数据库, 只包含三蛋白质的兴趣。细胞膜 MS 样品中含有三种毒素成分的肽, 分别为 LFN、PA 和 CMG2 的60.46%、67.97% 和54.15% 覆盖率 (图 7)。细胞膜后的结果, 如图 8所示, 仅含有来自 LFN和 PA (分别为57.41% 和67.79% 的覆盖率) 的肽。细胞膜后样品中 CMG2 的缺乏与孔隙形成过程中观察到的 BLI 纳米下降相一致。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57902/57902fig1.jpg"> 图 1.用 EM 和 MS 对 BLI 生物传感器组装和分离的蛋白质复合物进行分析的示意图.<a href="https://www.jove.com/files/ftp_upload/57902/57902fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57902/57902fig2.jpg"> 图 2.生物传感器活化: BLI 生物传感器定向特定组件的第一步。E126C 致命因子 n 终端域, (ifN) 是通过一个硫链链接, 创建适当的定向 PAprepore绑定接口。<a href="https://www.jove.com/files/ftp_upload/57902/57902fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57902/57902fig3.jpg"> 图 3.用 BLI 监测炭疽毒素的组装和拆卸: sensogram 显示, 由于炭疽毒素成分添加到生物传感器 (步骤4中) 时, 振幅的变化是时间的函数.表 1)。然后将 PAprepore加载到表面, 随后加入可溶性 CMG2 受体。所装配的预细胞膜复合体由一个 LFprepore-CMG2 组成。为了在晚期 endosome 环境中取得进展, 整个组装功能性炭疽毒素受到低 ph 脉 (ph 5.0) 的削弱, 使受体结合, 允许 prepore 过渡到其扩展膜插入孔构象。通过加入胶束, 确认和可溶性膜生成孔的暴露。<a href="https://www.jove.com/files/ftp_upload/57902/57902fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57902/57902fig4.jpg"> 图 4.BLI sensogram 的 CMG2 释放: 酸化步骤的痕迹显示初始增加或 "穗" 的振幅, 其次是较大的振幅下降, 可能是孔隙形成和受体离解, 分别。垂直虚线红色线表示新步骤的开始。Sensograms 在粗体虚线红色线上对齐。<a href="https://www.jove.com/files/ftp_upload/57902/57902fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57902/57902fig5.jpg"> 图 5.用 EM 和 ms 分析 BLI 生物传感器组装和分离的蛋白质复合物: 生物传感器附着的复合物很容易被释放成5µL 的缓冲, 其中包含了反色 EM 的可视化和 ms 识别.<a href="https://www.jove.com/files/ftp_upload/57902/57902fig5large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57902/57902fig6.jpg"> 图 6.与 EM 蛋白配合物的可视化: 预细胞膜样品网格, 显示密度与完整的三元配合物组成的 LFprepore-CMG2。后细胞膜复杂网格, 显示 PA 过渡到孔和可溶性的胶束, 没有明显的 CMG2 密度。预测复合体 (左手边) 的模型与所示的单个粒子的尺度相同。用预测蛋白 (根据 EM 密度的大小) 着色的粒子显示在每个粒子下面。<a href="https://www.jove.com/files/ftp_upload/57902/57902fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57902/57902fig7.jpg"> 图 7.蛋白质配合物与 ms 的验证: 预细胞膜 ms 样品包含所有三毒素组分的肽从 60.46%, 67.97% 和54.15% 覆盖率为 LFN, PA 和 CMG2, 分别。在错误发现率 (罗斯福) 中检测到的肽等于或高于 5%, 以黄色、罗斯福相等或高于1% 显示为绿色。<a href="https://www.jove.com/files/ftp_upload/57902/57902fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57902/57902fig8.jpg"> 图 8.蛋白质配合物的验证与 MS: 后细胞膜样品含有肽从 LFN和 PA (分别57.41% 和67.79% 覆盖), 但不 CMG2。罗斯福等于或高于5% 显示的黄色, 罗斯福等于或高于1% 显示绿色。<a href="https://www.jove.com/files/ftp_upload/57902/57902fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>步</td>
			<td>时间 (s)</td>
			<td>描述</td>
		</tr>
<tr>
<td>1</td>
			<td>30</td>
			<td>初始基线</td>
		</tr>
<tr>
<td>2</td>
			<td>420</td>
			<td>激活</td>
		</tr>
<tr>
<td>3</td>
			<td>300</td>
			<td>激活</td>
		</tr>
<tr>
<td>4</td>
			<td>300</td>
			<td>如果N加载</td>
		</tr>
<tr>
<td>5</td>
			<td>240</td>
			<td>半胱氨酸淬火</td>
		</tr>
<tr>
<td>6</td>
			<td>120</td>
			<td>基线</td>
		</tr>
<tr>
<td>7</td>
			<td>300</td>
			<td>PAprepore协会</td>
		</tr>
<tr>
<td>8</td>
			<td>30</td>
			<td>基线</td>
		</tr>
<tr>
<td>9</td>
			<td>300</td>
			<td>CMG2 协会</td>
		</tr>
<tr>
<td>10</td>
			<td>30</td>
			<td>基线</td>
		</tr>
<tr>
<td>11</td>
			<td>300</td>
			<td>酸性过渡</td>
		</tr>
<tr>
<td>12</td>
			<td>30</td>
			<td>基线</td>
		</tr>
<tr>
<td>13</td>
			<td>300</td>
			<td>胶束协会</td>
		</tr>
</tbody></table>表 1.单 BLI 运行到组装炭疽毒素复合物的步骤时间。请注意, 基线用于从上一步中冲洗掉未绑定的蛋白质和/或建立新的缓冲基线。

Watch this Scientific Journal Video about Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy at JoVE.c

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

在这里, 我们提出了一个协议, 以监测炭疽毒素的组装和拆卸使用 biolayer 干涉测量 (BLI)。在生物传感器表面组装/拆卸后, 通过电子显微镜和质谱法, 从表面释放出大量的蛋白质复合物, 用于可视化和识别复合物的成分。

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 ...

analyzing-dynamic-protein-complexes-assembled-on-released-from

Research

JoVE Journal

Biochemistry

9.3K 次观看.  University of Kansas Medical Center. 在这里, 我们提出了一个协议, 以监测炭疽毒素的组装和拆卸使用 biolayer 干涉测量 (BLI)。在生物传感器表面组装/拆卸后, 通过电子显微镜和质谱法, 从表面释放出大量的蛋白质复合物, 用于可视化和识别复合物的成分。

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

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

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 ...

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 ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

综合结构质谱分析蛋白质结构和蛋白质配体配合物

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

化学

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 ...

生物学

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T波离子迁移质谱蛋白复合物分析的基本实验程序

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

使用微流体扩展质量亮度分析浓度范围

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

作者聚焦：使用 MassFluidix 技术表征溶液中低亲和力蛋白质相互作用

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...

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 ...

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

摘要

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此视频中的章节

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

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

通过低温BN-MS分析进行高分辨率复杂分析

综合结构质谱分析蛋白质结构和蛋白质配体配合物

大蛋白复合物结构质谱分析

T波离子迁移质谱蛋白复合物分析的基本实验程序

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

蛋白复合物的鉴定