我们谨向 Willmitzer 博士表示感谢, 感谢他参与了该项目, 进行了富有成效的讨论, 并进行了重大监督。我们感谢丹尼尔 Veyel 博士对蛋白质组 MS 测量的帮助。我们感谢Änne 米氏夫人, 他为我们提供了宝贵的技术帮助与 LC MS 测量。此外, 我们还要感谢莫妮卡 Kosmacz 博士和 Ewelina 博士 Sokołowska 对原手稿工作的帮助和参与, 并向维罗尼卡 Jasińska 提供技术支持。

制备转基因拟南芥细胞培养线, 包括克隆、转化、选择和生长条件, 可在17中找到。请注意, EV 控制线建议纠正误报。在实验之前, 通过西方印迹分析确认诱饵蛋白的过度表达,例如对串联亲和标记的 g 蛋白部分使用 IgG 抗体。从植物细胞培养材料中分离生长培养基是很重要的。
1. 实验前制备植物细胞材料
<ol><li>生长一个 overexpressing 细胞培养线, 对18的蛋白质感兴趣。<ol>
<li>制备 MSMO 培养基, 其中含有4.43 克/升 MSMO 混合30克/升蔗糖。将缓冲液的 pH 值调整为 5.7, 用1米 KOH 和高压釜解决。在实验之前, 补充培养基与0.5 毫克/升α萘乙酸酸, 0.05 毫克/升激动和50微克/毫升卡那霉素。</li>
		<li>在一个100毫升的50毫升的 MSMO 培养基上, 用柔和的搅拌 (130 转每分钟), 在一个轨道平台上培养转化的植物细胞培养物。在20摄氏度的培养室中生长细胞, 光照强度等于80μmol m-2 s-1。</li>
		<li>亚文化细胞每7天进入新鲜培养基, 稀释1:10。</li>
	</ol>
</li>
	<li>在对数生长阶段收集细胞, 使用玻璃漏斗和真空泵结合使用尼龙网作为过滤器。包裹在铝箔中的渗透和冻结液氮。 注意: 请记住液氮非常冷。不正确的处理会导致烧伤。佩戴适当的个人防护设备, 包括隔热手套、防护眼镜和实验室大衣。</li>
</ol>2. 点击协议
注: 以下步骤从 Maeda等201411和 Van Leene等201117适应。
<ol><li>融汇收获和冷冻植物细胞培养材料使用混合机 (2 分钟在20赫兹) 或砂浆和杵, 以获得细粉。整除3克的地面材料 (相当于大约90毫克的总蛋白) 每样。使用液氮预冷设备, 避免在这一步骤中解冻样品。 注: 贮存地面植物材料在50毫升管在–80°c 到 AP 程序的开始。</li>
	<li>Triturate 在液-氮-预换热器砂浆中, 用3毫升冰冷裂解缓冲剂 (0.025 米 Tris–HCl pH 7.5; 0.5 米氯化钠; 1.5 毫米氯化镁2; 0.5 毫米; 1 毫米 NaF; 1 毫米 Na3); 100x 稀释的商业蛋白酶抑制剂鸡尾酒; 4 毫米PMSF) 直到物质融化。一旦样品解冻, 立即进行下一步。 注: 准备溶解缓冲液新鲜。在此步骤中引入空白示例。不推荐使用洗涤剂, 因为它们会导致 MS 检测中出现问题。</li>
	<li>要去除细胞碎片, 将材料分成2毫升离心管和离心机, 在 20817 x 克10分钟4摄氏度。在15毫升锥形离心管中收集3毫升的透明裂解液。</li>
	<li>在离心过程中, 平衡 IgG-琼脂糖珠。整除100µL 的每样珠子, 并用1毫升的裂解缓冲液清洗。涡并用重悬珠和闪光旋转。放弃溶解缓冲, 重复步骤两次。并用重悬珠在400µL 的裂解缓冲液中。</li>
	<li>在收集的植物裂解物中添加珠子, 在旋转轮上孵化混合物1小时, 在4摄氏度。</li>
	<li>将混合物通过鲁尔锁帽与具有过滤孔径35µm 的自旋柱一起转移到注射器中. 施加压力通过裂解液。带附着配合物的珠子将留在过滤器上, 而裂解液会经过。 注: 可选, 使用真空歧管系统。一定要施加温和的压力, 以免伤害珠子。</li>
	<li>先用10毫升洗涤缓冲器 (0.025 米 Tris–HCl ph 7.5; 0.5 米氯化钠), 再用1毫升洗脱缓冲器 (10 毫米 Tris–HCl pH 7.5; 150 毫米氯化钠; 0.5 毫米 EDTA; 1000x 稀释 E64 和1毫米 PMSF)。使用连接到柱或真空歧管系统的注射器进行洗涤。 注: 使用真空歧管系统时, 一定要施加温和的压力, 以免损害珠子。</li>
	<li>孵化珠与400µL 洗脱缓冲器包含 50 U 的改进版本的烟草蚀刻病毒 (AcTEV) 蛋白酶。使用 1000 rpm 的表振动筛, 30 分钟16摄氏度。 注: 请记住使用插头关闭底部的列添加洗脱缓冲。</li>
	<li>添加一个额外的部分 (50 U) 的酶入柱和孵化的混合物, 为未来30分钟在相同的, 上面描述的条件。</li>
	<li>通过离心 (1 分钟, 20817 x g) 或真空歧管收集2毫升离心管中的洗脱液。要去除剩余的配合物, 引入一个额外的洗脱步骤使用200µL 洗脱缓冲。 注: 将样品贮存在–20°c 或–80°c, 或立即进行蛋白质和代谢物萃取步骤。在冰上解冻冰冻样品。</li>
</ol>3. 西方污点分析
<ol><li>为了确认在收集的洗脱液中存在诱饵蛋白, 使用10µL 的 protein–metabolite 洗脱液进行 SDS–PAGE 和印迹分析。为了确定感兴趣的蛋白质, 使用小鼠主要抗体抗链亲和素结合蛋白 (1:200), TEV 蛋白酶解切后剩余的抽头标记的一部分, 如 Van Leene等201117所述。其次, 使用二次山羊抗鼠抗体与 HRP 结合。</li>
</ol>4. 代谢物和蛋白质提取
注: 本协议适用于 Giavalisco等201116。
注意: 从这一步开始使用 UPLC–MS–grade 解决方案。
<ol><li>将1毫升的甲基叔丁基醚 (MTBE)/甲醇/水溶剂 (3:1: 1) 添加到所收集的洗脱液中, 并通过反转来混合样品。确保溶剂在萃取步骤之前冷却到–20°c。 注意: 甲基叔丁基醚和甲醇是有害物质。在油烟机下执行萃取步骤, 并佩戴适当的个人防护设备,如手套。</li>
	<li>加入0.4 毫升甲醇: 水1:3 溶液对每个样品和混合样品的内容通过倒置。 注: 用甲醇补充混合物: 水溶液导致相分离。上部阶段包含脂质, 较低的阶段包含极性和半极性代谢物, 并且蛋白质在药丸能被发现。</li>
	<li>分离阶段, 离心样品在 20817 x g 在室温下2分钟, 然后收集上阶段的脂质测量 (没有在本协议中) 使用手动液体处理吸管与1毫升容积容量。</li>
	<li>加入0.2 毫升甲醇, 并通过反转混合。</li>
	<li>离心样品在 20817 x g 在 RT 2 分钟, 然后收集极性阶段为代谢物测量 (极性和半极性化合物)。为了避免干扰蛋白颗粒, 在管的底部留下大约50µL 的液相。</li>
	<li>在离心蒸发器中过夜的代谢物测量的干收集样品。避免在30–60分钟后从蒸发器中取出样品, 以免过度干燥蛋白颗粒。 注: 贮存样品在–20°c 或–80°c, 或立即着手准备蛋白质的 LC–MS/MS 分析。</li>
</ol>5. 为蛋白质组分析准备样品
注意: 这一步是由奥尔森等。200419和胰蛋白酶/赖氨酸 C 混合的技术手册 (见材料表)。
<ol><li>对样品进行酶消化。 注意: 在酶消化和样品脱盐过程中使用的溶剂是有害的。在油烟机下工作, 佩戴适当的个人防护设备,如手套。<ol>
<li>在30µL 的新制备变性缓冲液中溶解蛋白颗粒 (40 毫米含2米 thiourea/6 尿素的碳酸铵, pH 为 8)。为了达到更好的蛋白质溶解度, 执行15分钟超声波步骤。重复步骤, 直到颗粒溶解。</li>
		<li>离心样品在 20817 x g 10 分钟在4°c, 然后转移上清到一个新的离心管。</li>
		<li>用布拉德福德蛋白测定法测定蛋白质浓度。</li>
		<li>为进一步分析, 整除的体积相当于100µg 的蛋白质, 并填充样本多达46µL 变性缓冲。</li>
		<li>加入2µL 的新制备的还原缓冲剂 (50 毫米在 H2O 中溶解), 室温下孵育30分钟。</li>
		<li>用2µL 的新制备的烷基化反应缓冲剂 (150 毫米 iodoacetamide 溶解在40毫米的碳酸铵缓冲液中), 在室温下将混合物在黑暗中孵化20分钟。</li>
		<li>稀释样品与30µL 40 毫米碳酸氢铵缓冲和增加20µL LysC/胰蛋白酶混合物。</li>
		<li>4小时后孵化37摄氏度, 稀释样品与300µL 40 毫米碳酸氢铵缓冲。</li>
		<li>继续在37摄氏度的夜间孵化。</li>
		<li>酸化样品与大约20µL 10% 三氟乙酸酸 (TFA) 获得 pH 值 < 2。使用 ph 条检查样品 ph 值。 注: 将样品存储在–20°c 或继续下一步。</li>
	</ol>
</li>
	<li>淡化被消化的蛋白质。 注: 最好使用真空歧管系统。避免列过度干燥。<ol>
<li>冲洗 C18 SPE 柱 (见材料表) 与1毫升100% 甲醇, 然后1毫升80% 乙腈 (ACN) 含 0.1% TFA 稀释在水中。使用, 在这里和进一步的蛋白质脱盐步骤, 一个真空流形系统, 以加快进程。避免列过度干燥。</li>
		<li>用1毫升的 0.1% TFA 稀释在水中, 平衡该柱。</li>
		<li>将该示例加载到该列上。冲洗管与额外的200µL 0.1% TFA 和转移的解决方案的列。通过该列运行解决方案。</li>
		<li>用1毫升 0.1% TFA 洗两次柱。</li>
	</ol>
</li>
	<li>洗脱淡化肽从柱800µL 60% ACN, 0.1% TFA 溶液。在离心蒸发器中干燥所收集的分数, 避免在30–60后从蒸发器中取出样品, 以免蛋白质组分过度干燥。 注: 在–20°c 或–80°c 储存样品或立即进行下一步。在随后的步骤中, 将样品放在冰上。</li>
</ol>6. 使用 UPLC–MS/MS 测定制备的蛋白质样品。
注: 在蛋白质组和 metabolomic 测量之前, 过滤器 (0.2 µm 孔大小) 和德加德所有缓冲器使用真空泵为1小时。
<ol><li>并用重悬干肽颗粒储存在2毫升离心管在50µL 的缓冲 C (3% 伏/五 ACN, 0.1% v/v 甲酸) 使用手动液体处理吸管与200µL 容积容量。油脂实验样品15分钟, 在超声浴与35赫超声波频率。 注意: ACN 和甲酸是有害物质。在油烟机下工作, 佩戴适当的个人防护设备,如手套。</li>
	<li>离心样品在 20817 x g 10 分钟在4°c, 然后转移20µL 的上清到玻璃瓶。</li>
	<li>用 C18 反相柱连接到液相色谱分离消化肽, 利用质谱仪获得质谱。<ol>
<li>使用 300-µL/分钟流量, 在样本的3列上分离。对于移动阶段, 使用缓冲 C 和 D (63% 伏/v ACN, 0.1% 伏/v 甲酸), 形成梯度从 3% ACN 到 15% ACN 超过20分钟, 然后到 30% ACN 在未来10分钟。 注: 将样品的其余部分保存在–20°c 或–80°c, 最多数月。在蛋白质组测量之前, 冻结在冰上的样品。</li>
		<li>用 60% ACN 清洗污染物10分钟, 平衡5µL 的列, 然后再测量下一个样品。</li>
		<li>利用数据依赖的 ms/毫秒方法获得质量谱, 分辨率设置为 7万, 3e6离子的 AGC 目标, 最大注入时间为100毫秒, 从300到1600不等的 m/z。获得最大15毫秒/毫秒扫描分辨率为 17500, AGC 目标为 1e5, 最大注入时间为100毫秒, 底充胶比率为 20%, 与隔离窗口1.6 米/z 和 m/z 不等, 从200到2000。启用顶点触发器 (6–20 s), 将动态排除设置为十五年代, 并排除1和 > 5 的费用。</li>
	</ol>
</li>
</ol>7. 蛋白质组数据的处理
<ol><li>从 http://www.uniprot.org/下载最新拟南芥蛋白质组数据库, 包括污染物数据库。分析从 LC–MS 运行获得的原始数据使用 MaxQuant 与集成的仙女座肽搜索引擎使用默认设置与启用 LFQ 正常化20,21,22。查找有关表 S1中使用的参数的详细信息。</li>
	<li>打开 "蛋白质 groups.txt" 输出文件。为了进一步分析, 筛选出至少有两个独特的肽的蛋白质组。删除由 MaxQuant 定义的蛋白质组作为潜在的污染物和筛选的. 芥蛋白 (ARATH 在 Fasta 头专栏) 存在于数据库中。</li>
	<li>为了检验样品间蛋白质富集的意义, 使用 LFQ 规范化强度, 进行配对, 两尾学生的 t 检验, 然后进行多重比较校正 (如Benjamini & 霍赫贝格假发现率 (罗斯福)更正或 Bonferroni 更正)。<ol>
<li>通过比较获得的 EV 控制和 NDPK1 的 LFQ 强度计算 p 值。筛选出所有待定值。按升序对 p 值进行排序, 并使用 R 脚本或在线计算器 (如https://www.sdmproject.com/utilities/？show=FDR) 计算罗斯福更正。筛选罗斯福价值低于0.1。 注: 考虑数据分析的形式适合于研究。定量研究 (样品间蛋白质富集分析) 使用 "LFQ 强度" 值, 而对于定性研究 (存在或缺乏特定的蛋白质) 选择 "强度" 值。</li>
		<li>筛选 NDPK1 比 EV 控制更丰富的蛋白质组。确定潜在的蛋白质合作伙伴的本地化使用 SUBA 数据库23和正确的蛋白质与 NDPK1 的共同本地化。</li>
	</ol>
</li>
</ol>8. 使用 UPLC–MS 测量含有极性相的样品。
<ol><li>并用重悬干燥极性阶段从步骤4.5 在200µL 水和油脂实验样品为5分钟。</li>
	<li>离心样品在 20817 x g 10 分钟在4°c, 然后转移上清到玻璃瓶。 注: 将样品的其余部分储存在–20°c 或–80°c, 可达数月。在 metabolomic 测量之前, 冻结冰上的样品。</li>
	<li>使用 UPLC 耦合 C18 反相柱进行分离步骤, 并获得质谱与 MS。<ol>
<li>将每个电离模式 (正和负) 上的每个注入样品的2µL 列加载到该柱上, 并使用400µL/最小流速分隔分数。要创建代谢物测量所需的梯度, 请准备移动相解, 如下所示: 缓冲 A (0.1% 甲酸在 H2O) 和缓冲 B (0.1% 甲酸在 ACN)。</li>
		<li>分离代谢物在400µL/分钟和以下梯度: 1 分钟99% 缓冲 a, 11 分钟线性梯度从99% 缓冲 a 到60% 缓冲 a, 13 min 线性梯度从60% 缓冲 a 到30% 缓冲 a, 15 min 线性梯度从30% 缓冲 a 到1%缓冲 a, 保持1% 浓度, 直到16分钟从17分钟开始, 使用线性渐变从1% 缓冲区 a 到99% 缓冲区 a. 重新平衡列为3分钟与99% 集中缓冲 a 在测量下一个样品之前。</li>
		<li>获得100至1500米/z 的质量谱, 分辨率设置为 2.5万, 加载时间限制为100毫秒. 设置 AGC 目标为 1e6, 毛细管电压为 3kV, 带鞘气流量和辅助气体值60和20分别。将毛细管温度设置为250°c 和撇嘴电压为25V。</li>
	</ol>
</li>
</ol>9. 新陈代谢数据的处理
<ol><li>从两种电离模式中采集的图谱。使用软件提取质量的电荷比 (m/z), 保留时间 (RT) 和强度的相关峰值,例如,商业软件 (见材料表) 或备选案文24。<ol>
<li>通过双击 .exe 文件开始处理软件</li>
		<li>创建新工作流, 搜索活动 "从文件加载" 并将此活动通过 "拖放" 移动到空白工作流空间中。用鼠标右键按活动并打开活动设置。<ol>
<li>在包含 "常规" 设置的选项卡中, 在 "名称" 字段中设置实验名称, 然后单击 "选择文件和文件夹" 并标记原始图谱。</li>
			<li>在包含 "高级" 设置的选项卡中, 将 "配置文件数据截止" 设置为0强度。单击 "应用" 和 "确定"。</li>
		</ol>
</li>
		<li>搜索并添加活动 "数据扫描"。用鼠标右键按活动并打开活动设置。<ol>
<li>在包含 "常规" 设置的选项卡中, 标记 "质心数据" 和 "ms/ms 数据"。通过在 "选择" 面板中选择 "全部", 删除所有 ms/毫秒数据。</li>
		</ol>
</li>
		<li>搜索并添加活动 "色谱化学噪声减法"。用鼠标右键按活动并打开活动设置。<ol>
<li>在包含 "常规" 设置的选项卡中, 标记 "色谱平滑", 并将扫描次数设置为 "3" 和 "估计值" 到 "移动平均值"。将 "RT 窗口" 设置为51扫描, "分量" 为 50%, 减法 "方法" 和750强度 "阈值"。</li>
			<li>在包含 "高级" 设置的选项卡中, 标记 "rt 结构删除", 并将 "最小 rt 长度" 设置为5扫描。</li>
			<li>在包含 "高级" 设置的选项卡中, 标记 "m/z 结构删除", 并将 "最小 m-/z 长度" 设置为3磅。</li>
		</ol>
</li>
		<li>搜索并添加活动 "色谱 RT 对齐"。用鼠标右键按活动并打开活动设置。<ol>
<li>在包含 "常规" 设置的选项卡中, 将 "校准方案" 设置为 "对齐基准树" 和 "RT 搜索间隔", 以0.5 分钟为准。</li>
			<li>在包含 "高级" 设置的选项卡中, 使用默认参数。</li>
		</ol>
</li>
		<li>搜索并添加 "色谱" 组活动中的 "峰值检测" 活动。用鼠标右键按活动并打开活动设置。<ol>
<li>在包含 "常规" 设置的选项卡中, 将 "求和窗口" 设置为0.09 分钟, "最小峰值大小" 为0.03 分钟, "最大合并距离" 为5磅, "合并策略" 为 "中心"。在 "峰值 RT 分裂" 框设置 "间隙/峰值比率" 到50%。</li>
			<li>在包含 "高级" 设置的选项卡中, 将 "平滑窗口" 设置为5磅, "细化阈值" 为80% 和 "一致性阈值" 为1。设置 "中心计算" 为 "强度加权" 的 "强度阈值" 设置为70%。</li>
		</ol>
</li>
		<li>在 "色谱" 活动组中搜索并添加活动 "同位素聚类"。用鼠标右键按活动并打开活动设置。<ol>
<li>在包含 "常规" 设置的选项卡中, 将 "RT 公差" 设置为0.015 分钟和 "m/z 公差" 为 5 ppm。</li>
			<li>在包含 "信封管接头" 设置的选项卡中, 将 "方法" 设置为 "无形状限制" 和 "电离" 为 "质子 (为正模式)" 和 "Deprotonation" (用于负模式)。分别将 "最小和最大电荷" 设置为1和4。</li>
			<li>在包含 "高级" 设置的选项卡中, 使用默认参数。</li>
		</ol>
</li>
		<li>搜索并添加活动 "单一筛选器"。</li>
		<li>要导出数据处理的结果, 请在 "导出" 活动组中搜索并添加活动 "分析师"。<ol>
<li>在包含 "常规" 设置的选项卡中, 将 "类型" 设置为 "簇", "可观察" 为 "汇总强度"。选择 "自定义目标" 并指定导出文件的目录。</li>
			<li>在包含 "高级" 设置的选项卡中, 使用默认参数。</li>
		</ol>
</li>
	</ol>
</li>
	<li>使用内部引用复合数据库对质量特征进行注释。<ol>
<li>用 UPLC-ms 分析一个或多个 MS 级参考化合物. 使用相同的 LC-ms 方法分析参考化合物和代谢产物, 并与感兴趣的蛋白质进行共纯化。 注: 本研究将一组近300合成二肽进行分析, 作为参考复合库。</li>
		<li>使用分析 (见材料表) 软件打开原始色谱文件, 并搜索特定的 m/z 和 RT 相关的测量参照化合物 (见用户指南)。 注: 次生代谢物在电离类型上有差异。通过搜索等于 M-1.007276、m + 1.007276、m + 18.033823 和 m + 22.989218 (m h)、[m + H]、[m + NH4] 和 [m + Na] 的离子质量, 检查常见加合物是否存在。</li>
		<li>使用电子表格打开导出的 "分析" 文件, 在处理图谱后获得, 并搜索特定的离子质量。比较实验中测量的质量特征和参考化合物的 rt。允许偏差 0.005 Da 为 m/z 和0.1 分钟的 RT。</li>
	</ol>
</li>
	<li>为了检验在样品 (抗原蛋白与 EV 控制) 之间用特定蛋白质对代谢产物进行富集共纯化的意义, 用两尾非配对学生的 t 检验来比较峰值值, 后跟多个比较校正 (例如Benjamini & 霍赫贝格错误发现率更正或 Bonferroni 更正)。</li>
</ol>

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作者没有什么可透露的。

所提出的协议允许平行识别靶蛋白的 PP 和 PM 复合物。从克隆到最终结果, 实验可以在8-12 周内完成。完整 AP 需要大约4-6 小时的12到24个样本, 使我们的协议适用于中吞吐量分析。
尽管该议定书总体上直截了当, 但仍有一些关键步骤。(i) 足够数量的输入蛋白和亲和珠对于达到动态的代谢产物检测至关重要。因此, 有效的细胞裂解是过程中的关键步骤。不良的蛋白质产量可能是由于材料的粉碎不足或次优裂解-缓冲/物质比的结果。(二) 应注意使用的试剂是 MS 友好的。在干扰 MS 检测时, 应避免使用强力洗涤剂、甘油或过量食盐。(iii) 在洗涤步骤中, 琼脂糖珠不应过度干燥, 当使用真空歧管时, 应用慢速流速, 以免破坏珠子或影响复杂的稳定性是很重要的。
对所提出的协议有一些重要的可能的修改: (i) 我们使用本构 CaMV35S 启动子来最大化诱饵蛋白的数量。过度表达虽然非常有用, 但会对细胞稳态28产生严重影响, 导致生理上无关的相互作用的形成。标记蛋白的表达使用本地启动器, 并在可能的功能损失的背景被认为优于检索真正的生物 interactors。对于通常没有在植物细胞培养中表达的蛋白质, 植物的背景可能证明有必要确定相关的 interactors。(ii) 在使用膜蛋白时, 溶解缓冲液需要辅以 MS 兼容洗涤剂。(iii) 引入第二亲和纯化步骤可提高误报率, 并消除对 EV 控制29的需要。一个新的串联标签与两个独立的蛋白酶解的网站提出了一个有吸引力的替代的大小排除色谱步骤添加的 Maeda等201411, 这既费力又费时。
AP 最严重的缺点是误报率高。原因很多。本构表达已被提及。另一种生理上无关的相互作用的来源, 除非与孤立的细胞器, 是制备全细胞裂解物包含蛋白质和代谢物的不同亚细胞间的混合物。亚细胞定位应用于筛选真正的 interactors。然而, 大多数假阳性是由蛋白质和琼脂糖树脂之间不特异的结合引起的。如上文所述, 引入第二个净化步骤提供了解决问题的最佳方法, 但代价是时间和吞吐量。此外, 随着协议的延长, 较弱的交互可能会丢失。AP 的另一个告诫是, 尽管它提供了关于目标蛋白 interactome 的全面信息, 但在诱饵蛋白的直接和间接目标之间进行区分是不可能的。需要有针对性的分子方法来确认交互。
采用 AP 结合 MS 为基础的新陈代谢用于研究12的酵母蛋白复合物.这项工作, 连同我们早先的观察13 , 类似于脂质, 极性和半极性化合物仍然绑定到从细胞裂解物分离的蛋白质复合物, 为提出的议定书提供了概念基础。我们的协议的特点是三独特的点: (i) 与酵母工作12相比, 它表明, AP 是适合检索不仅疏水性, 而且亲水蛋白配体。(ii) 通过引入一个三入一萃取协议, 单一 AP 可以用来研究蛋白和代谢物 interactors 的诱饵蛋白。(iii) 我们修改了该议定书以种植细胞。
未来的努力将集中在创建一个新的串联标签与两个独立的蛋白酶解的网站。我们还想探讨该议定书对低丰度小分子如植物激素的适用性。

此处描述的方法旨在确定在体内细胞裂解条件下选择蛋白质的代谢物和蛋白质伙伴。据推测, 今天有许多比特征的代谢物具有重要的调节功能1。代谢物可以充当生物开关, 改变其受体蛋白2、3、4的活动、功能和/或定位。在过去十年中, 已经制定了一些突破性的方法, 能够在体内或近体条件下鉴定 PMI, 已经发展了5。可用方法可分为两组。第一组包括从已知代谢物诱饵开始的技术, 以诱捕新的蛋白质伙伴。方法包括亲和层析6、药物亲和反应靶-稳定性测定7、化疗蛋白质组学8和热蛋白质分析9。第二组由一个单一的方法, 开始与已知的蛋白质, 以确定小分子配体10,11。
采用 AP 结合 MS 为基础的 lipidomics 分析酵母蛋白脂复合物12.作为出发点, 作者使用酵母菌株表达21种酶参与麦角甾醇生物合成和103激酶融合到串联亲和纯化 (自来水) 标签。70% 的酶和20% 的激酶被发现绑定不同的疏水性配体, 脱落光进入复杂的蛋白质-脂质相互作用网络。
以前, 我们可以证明, 类似于脂质, 极性和半极性化合物也仍然绑定到从细胞裂解物13分离的蛋白质复合物。根据这些发现, 我们决定优化以前发表的10、11植物细胞和亲水性化合物14的 AP 方法。为此, 我们使用了 Van Leene等2010 所描述的抽头矢量, 成功地用于植物 PPI 研究15。为了缩短获得转基因品系所需的时间, 我们决定了拟南芥细胞培养。我们使用一步甲基叔丁基醚, (MTBE)/甲醇/水萃取方法, 允许蛋白质 (颗粒), 脂类 (有机相) 和亲水性代谢物 (水相)16在一个单一的亲和纯化实验。EV 控制线被引入, 以排除误报,例如蛋白质绑定到标签单独。作为概念的证明, 我们标记了三 (五) 核苷二磷酸激酶存在于拟南芥基因组 (NDPK1-NDPK3)。在其他发现中, 我们可以证明 NDPK1 与谷胱甘肽S转移酶和谷胱甘肽相互作用。因此, 我们可以证明 NDPK1 受 glutathionylation14。
综上所述, 所提出的协议是描述蛋白质和蛋白质-小分子相互作用网络的重要工具, 是对现有方法的重大进展。

<table><tbody><tr><td>Murashige and Skoog Basal Salts with minimal organics</td><td>Sigma-Aldrich</td><td>M6899</td><td></td></tr><tr><td>1-Naphthylacetic acid</td><td>Sigma-Aldrich</td><td>N1641</td><td></td></tr><tr><td>Kinetin solution</td><td>Sigma-Aldrich</td><td>K3253</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>10708976001</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>MgCl2</td><td>Carl Roth</td><td>2189.1</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>3609</td><td></td></tr><tr><td>NaF</td><td>Sigma-Aldrich</td><td>S6776</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>E-64 protease inhibitor</td><td>Sigma-Aldrich</td><td>E3132</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>P9599</td><td></td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S6508</td><td></td></tr><tr><td>AcTEV Protease</td><td>Thermo Fischer Scientific</td><td>12575015</td><td></td></tr><tr><td>Rotiphorese&amp;nbsp;Gel 30 (37,5:1)</td><td>Carl Roth</td><td>3029.2</td><td></td></tr><tr><td>TEMED</td><td>Carl Roth</td><td>2367.3</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder</td><td>Thermo Fischer Scientific</td><td>26616</td><td></td></tr><tr><td>SBP Tag Antibody (SB19-C4)</td><td>Santa Cruz Biotechnology</td><td>sc-101595</td><td></td></tr><tr><td>Goat anti-mouse IgG-HRP</td><td>Santa Cruz Biotechnology</td><td>sc-2005</td><td></td></tr><tr><td>Bradford Reagent</td><td>Sigma-Aldrich</td><td>B6916</td><td></td></tr><tr><td>Trypsin/Lys-C Mix, Mass Spec Grade</td><td>Promega&amp;nbsp;</td><td>V5071</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5128</td><td></td></tr><tr><td>Thiourea</td><td>Sigma-Aldrich</td><td>T8656</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I1149</td><td></td></tr><tr><td>MTBE&amp;nbsp;</td><td>Biosolve</td><td>138906</td><td></td></tr><tr><td>Methanol&amp;nbsp;</td><td>Biosolve</td><td>136806</td><td></td></tr><tr><td>Water</td><td>Biosolve</td><td>232106</td><td></td></tr><tr><td>Acetonitrile</td><td>Biosolve</td><td>12006</td><td></td></tr><tr><td>Trifluoroacetic acid&amp;nbsp;</td><td>Biosolve</td><td>202341</td><td></td></tr><tr><td>Formic acid&amp;nbsp;</td><td>Biosolve</td><td>69141</td><td></td></tr><tr><td>Unimax 2010 Platform Shaker</td><td>Heidolph</td><td>5421002000</td><td></td></tr><tr><td>Nylon Mesh (Wire diameter 34 &amp;micro;M, thickness 55 &amp;micro;M, open area 14%)</td><td>Prosepa</td><td>Custom order</td><td></td></tr><tr><td>Glass Funnel, 47 mm, 300 ml</td><td>Restek</td><td>KT953751-0000</td><td></td></tr><tr><td>Filter Bottle Top 500 mL 0,2 &amp;micro;M Pes St</td><td>VWR International GmbH</td><td>514-0340</td><td></td></tr><tr><td>Mixer Mill MM 400</td><td>Retsch GmbH</td><td>207450001</td><td></td></tr><tr><td>IgG Sepharose 6 Fast Flow</td><td>GE Healthcare Life Sciences</td><td>17-0969-02</td><td></td></tr><tr><td>Mobicol &quot;&quot;Classic&quot;&quot; with 2 different screw caps without filters</td><td>MoBiTec GmbH</td><td>M1002</td><td></td></tr><tr><td>Filter (small) 35 &amp;micro;M pore size, for Mobicol M 1002, M1003, M1050 &amp;amp; M1053</td><td>MoBiTec GmbH</td><td>M513515</td><td></td></tr><tr><td>Variable Speed Tube Rotator SB 3</td><td>Carl Roth</td><td>Y550.1</td><td></td></tr><tr><td>Rotary dishes for rotators SB 3</td><td>Carl Roth</td><td>Y555.1</td><td></td></tr><tr><td>Resprep 24-Port SPE Manifolds</td><td>Restek&amp;nbsp;</td><td>26080</td><td></td></tr><tr><td>Finisterre C18/17% SPE Columns 100mg / 1ml</td><td>Teknokroma</td><td>TR-F034000</td><td></td></tr><tr><td>Autosampler&amp;nbsp;Vials</td><td>Klaus Trott Chromatographie-Zubeh&amp;ouml;r</td><td>40 11 01 740</td><td></td></tr><tr><td>Acclaim PepMap 100 C18 LC Column</td><td>Thermo Fischer Scientific</td><td>164534</td><td></td></tr><tr><td>EASY-nLC 1000 Liquid Chromatograph</td><td>Thermo Fischer Scientific</td><td>LC120</td><td></td></tr><tr><td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td><td>Thermo Fischer Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>Acquity UPLC system&amp;nbsp;</td><td>Waters&amp;nbsp;</td><td>Custom order</td><td></td></tr><tr><td>ACQUITY UPLC HSS C18 Column, 100A, 1.8 &amp;micro;M, 2.1 mM X 100 mM, 1/pkg</td><td>Waters&amp;nbsp;</td><td>186003533</td><td></td></tr><tr><td>High-power ultrasonic cleaning baths for aqueous cleaning solutions</td><td>Bandelin</td><td>RK 31</td><td></td></tr><tr><td>Genedata Expressionist</td><td>Genedata</td><td>NaN</td><td></td></tr><tr><td>Xcalibur Software</td><td>Thermo Fischer Scientific</td><td>NaN</td><td></td></tr><tr><td>MaxQuant</td><td>NaN</td><td>NaN</td><td></td></tr></tbody></table>

2 in 1: one-step affinity purification for the parallel analysis of protein-protein and protein-metabolite complexes

细胞过程由蛋白质、代谢物和核酸等生物分子之间的相互作用调节。虽然对蛋白质-蛋白质相互作用 (PPI) 的研究并不新奇, 但旨在表征内源性蛋白质代谢物相互作用 (PMI) 的实验方法构成了一个较近期的发展。在此, 我们提出一个协议, 允许同时描述 PPI 和 PMI 的蛋白质的选择, 称为诱饵。我们的协议优化了拟南芥细胞培养, 并结合亲和纯化 (AP) 与质谱 (MS) 为基础的蛋白质和代谢物检测。总之, 转基因拟南芥系, 表达诱饵蛋白融合到亲和标记, 首先裂解获得一个本地的细胞提取物。抗标记抗体是用来拉下蛋白质和代谢物的伙伴的诱饵蛋白。采用一步法甲基叔丁基醚 (MTBE)/甲醇/水法提取亲和纯化复合物。虽然代谢物分离成极性或疏水相, 蛋白质可以在颗粒中找到。然后用超效液相色谱-质谱 (UPLC-ms 或 UPLC-ms) 分析代谢产物和蛋白质。空矢量 (EV) 控制线用于排除误报。我们的协议的主要优点是, 它能够在近生理条件 (细胞裂解物) 中同时识别目标蛋白的蛋白质和代谢物伙伴。所提出的方法是直接的, 快速的, 可以很容易地适应生物系统以外的植物细胞培养。

在原研究中, 三A. NDPK 基因在抗原细胞悬浮培养物的控制下, 在本构35S 启动子14 (图 1) 下进行。串联亲和标记被融合到一个诱饵蛋白的羧基或氨基端端。亲和纯化复合物受叔丁基醚/甲醇/水萃取16。用 MS (表 S2 和 S3) 识别亲和拉蛋白和小分子。
为了纠正误报, 用空白样本排除了化学物质和实验室消耗品中的小分子污染物。此外, 使用 EV 控制线来计算与亲和标记或树脂单独结合的代谢物和蛋白质。为了检索真正的阳性, 双尾非配对学生的 t 检验和 Benjamini & 霍赫贝格假发现率校正被用于识别代谢物 (表 S4) 和蛋白质 (表 S5) 显著丰富的 NDPKs AP实验 (N 和 C 末期标记 NDPKs) 与 EV 控制线 (罗斯福 < 0.1) 比较。请注意, 在以前的工作中, 我们使用缺勤/存在标准来描绘蛋白质和小分子 interactors。
给出了 NDPK1 的代表性结果, 而代谢物数据集中于合成二肽, 这是本组研究的一种新型小分子调节器。蛋白质组分析揭示了 26 NDPK1 的蛋白质合作伙伴。通过进一步过滤蛋白质与 NDPK1 (细胞质) 在同一个亚细胞室中的联合定位, 该清单缩小到13假定的蛋白质 interactors。在鉴定的蛋白质中有谷胱甘肽S转移酶、两个伸长起始因子、蛋白和 aconitate 水合。Metabolomic 分析揭示了四合成二肽, 即谷、伊、列和洗脱与 NDPK1 (图 2)。请注意, 所有四合成二肽在其 n-端共用疏水性残渣, 表明具有共同的结合特异性。
为了寻找已知的蛋白质-蛋白质和蛋白质代谢物络合物, 我们查询了13识别的蛋白质和四合成二肽对针数据库25 (图 3)。可以提出几点意见: (一) interactors 以前没有 NDPK1 的报告。(ii) 报告 APX1 直系同源与醛脱氢酶家族成员 ALDH7B4 相互作用, 而翻译起始因子 FBR12 以基因 AT2G40290 编码的另一种转化起始因子。(三) 确定的合成二肽没有报告的蛋白质伙伴。洗脱合成二肽未被报告为与任何检索到的植物蛋白相关联。然而, 它们在其他生物体中扮演着重要的角色: 列伊岛,例如, 在人类细胞26行中具有神经营养因子激活作用。请注意, 该实验不允许识别系统的精确拓扑。例如, 二肽可能直接与 NDPK1 相互作用, 但很可能与任何共同纯化的蛋白质有关。
结合起来, 我们的研究结果表明, 建立的程序, 采用 AP 和质谱, 促进蛋白质蛋白和蛋白质-小分子 interactors 的鉴定, 并有助于产生广泛的信息,目标蛋白的 interactome。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57720/57720fig1.jpg"> 图1。AP-MS 工作流方案。(a)从植物细胞培养中制备本族可溶性分数。(B) AP 程序中的下一步步骤。将样品加载到柱上后, 将所附的兴趣蛋白 (点) 与一个分路标记结合, 并与固定在琼脂糖珠上的 IgG 抗体结合。洗涤柱有助于去除未绑定的蛋白质和代谢物。AcTEV 卵裂后, 洗脱蛋白代谢物复合体。(C)将络合物分离成蛋白质和代谢物分数, 然后进行半定量 MS 分析。这个数字的一部分是从 Luzarowski等201714转载。<a href="/files/ftp_upload/57720/57720fig1large.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/57720/57720fig2.jpg"> 图2。合成二肽专门与 NDPK1 共同洗脱.绘制了 AP 试验中测定的四合成二肽(A)、谷 ( B)、列伊岛(C)和(D)的平均强度。与 EV 控制 (星号代表罗斯福 < 0.1) 相比, 所有四合成二肽在 NDPK1 样品中均显着丰富。误差线表示6测量的标准错误 (3 复制 N 和3的 C 末期标记蛋白)。<a href="/files/ftp_upload/57720/57720fig2large.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/57720/57720fig3.jpg"> 图3。所有分子的相互作用网络与 NDPK1 共洗脱, 查询对缝合数据库仅考虑以前的实验和数据库证据 (信心 > 0.2).较高的置信度表明了较高的交互几率, 并根据沉积数据进行了计算。<a href="/files/ftp_upload/57720/57720fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
表 S1。MaxQuant 输出表 "parameters.txt"。表包括用于标识和量化的阈值, 以及有关所用数据库的信息。<a href="/files/ftp_upload/57720/Table_S1.xlsx">请单击此处下载此文件.</a>
表 S2。来自 MaxQuant 输出表 "proteinGroups.txt" 的信息。表包含所有已识别的蛋白质组、强度和其他信息 (如独特的多肽和分数) 的列表。<a href="/files/ftp_upload/57720/Table_S2.xlsx">请单击此处下载此文件.</a>
表 S3。含有极性代谢物分析的输出文件。表包含所有已识别的质量特征的列表, 其特征是特定的 m-/z、RT 和强度。<a href="/files/ftp_upload/57720/Table_S3.xlsx">请单击此处下载此文件.</a>
表 S4。合成二肽发现的 AP 样品中, NDPK1, NDPK2 或 NDPK3 被用作诱饵。合成二肽在空白样品中的显示被排除在名单之外。两条独立的线 (标记在 N 或 C 总站) 为每个 NDPK 的运行三个三。采用 Benjamini & 霍赫贝格法对学生进行t检验并对p值进行进一步修正, 以确定 NDPKs (罗斯福 < 0.1) 显著丰富的 interactor 合作伙伴。给出的是ΔRT 计算与参考化合物和Δppm 有关的 monoisotopic 质量在 Metlin27。<a href="/files/ftp_upload/57720/Table_S4.xlsx">请单击此处下载此文件.</a>
表 S5。蛋白质与 NDPK1 共纯化。两条独立的线 (标记在 N 或 C 总站) 为每个 NDPK 的运行三个三。采用 Benjamini & 霍赫贝格法对学生进行t检验并对p值进行进一步修正, 以确定 NDPKs (罗斯福 < 0.1) 显著丰富的 interactor 合作伙伴。<a href="/files/ftp_upload/57720/Table_S5.xlsx">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about 2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes at JoVE.com

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

蛋白质和蛋白质代谢物相互作用对所有细胞功能都至关重要。在这里, 我们描述了一个协议, 允许并行分析这些交互与蛋白质的选择。我们的协议优化了植物细胞培养, 并结合亲和纯化与质谱为基础的蛋白质和代谢物检测。

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

2-1-one-step-affinity-purification-for-parallel-analysis-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

11.2K 次观看.  Max Planck Institute of Molecular Plant Physiology. 蛋白质和蛋白质代谢物相互作用对所有细胞功能都至关重要。在这里, 我们描述了一个协议, 允许并行分析这些交互与蛋白质的选择。我们的协议优化了植物细胞培养, 并结合亲和纯化与质谱为基础的蛋白质和代谢物检测。

视频: 2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

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

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

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 ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

使用多柱板适配器的经济性和多功能性高通量蛋白质净化系统

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

<meta charset="utf8"></head><body>优化 FPLC 以高效纯化 His 标记的 FEN1 蛋白

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

<meta charset="utf8"></head><body>作者聚焦:优化 His 标记的 FEN1 蛋白的亲和层析

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化 

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

生物学

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 ...

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

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此视频中的章节

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

从真核细胞蛋白质复合体串联亲和纯化

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

使用多柱板适配器的经济性和多功能性高通量蛋白质净化系统

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

蛋白复合物的鉴定

在EMBL的MultiBac蛋白质复杂的生产平台