我们感谢细胞骨架 Inc. 研究科学家和 Drs. 布莱恩. 胡佛和阿什利. 戴维斯对手稿进行评论、编辑和富有成效的讨论。此外, 我们感谢 Dr. 在制作视频手稿时所作的贡献。这项工作得到了资助, 从骨架 Inc。

1. 样品制备: 细胞培养
<ol><li>在 Dulbecco 氏改良的鹰培养基 (DMEM) 中生长四150毫米的 A431 细胞板, 补充10% 胎牛血清。<ol>
<li>将 A431 细胞生长到 50% 70-100。</li>
		<li>血清对24小时无血清 DMEM 的四板 A431 细胞的限制。</li>
		<li>用33.3 毫克/毫升的表皮生长因子治疗两个 A431 细胞, 1 小时, 其余两个板块不予处理。 注: 此处显示的细胞生长和治疗条件提供了一个例子;然而, 这种方法适用于许多不同的细胞类型和治疗条件。</li>
	</ol>
</li>
	<li>用抑制剂制备 blastR 裂解和稀释缓冲液 (表 1)。结合2.895 毫升的 blastR 裂解缓冲液与15µL 酪氨酸磷酸酶抑制剂, 30 µL deubiquitinase 和 deSUMOylase 抑制剂, 30 µL 的组蛋白乙酰 (HDAC) 抑制剂, 和30µL 蛋白酶抑制剂鸡尾酒。<ol>
<li>结合9.65 毫升的 blastR 稀释缓冲器与50µL 酪氨酸磷酸酶抑制剂, 100 µL deubiquitinase 和 deSUMOylase 抑制剂, 100 µL 的组蛋白乙酰 (HDAC) 抑制剂, 和100µL 蛋白酶抑制剂鸡尾酒。 注意: 有关缓冲成分和抑制剂信息, 请参见材料表。</li>
	</ol>
</li>
	<li>倒掉培养基, 把细胞放在冰上。然后, 用10毫升的1x 磷酸缓冲盐水 (PBS) 冲洗两次细胞。<ol>
<li>删除尽可能多的 PBS 之前, 添加 blastR 裂解缓冲, 以最大限度地裂解细胞。 注意: 有关缓冲区组合, 请参见材料表。</li>
	</ol>
</li>
	<li>在每150毫米板上加入600µL 的 blastR 裂解缓冲液。缓冲区的数量基于预期的蛋白质产量 (表 2)。<ol>
<li>用刮板机溶解电池。裂解液会因核裂解而变得黏稠。</li>
	</ol>
</li>
	<li>使用一个剪1毫升吸管针尖转移到一个 blastR 过滤器已放置在一个15毫升收集管 (图 1) 的原油裂解。在这里, 使用15毫升猎鹰管。</li>
	<li>使用提供的过滤柱塞来完全压缩 blastR 过滤器, 并收集在15毫升管 (图 1) 中的裂解液, 包括任何气泡。</li>
	<li>可选: 转移澄清裂解至1.5 毫升离心管和离心裂解液在大约 1万 x g 1 分钟, 在4° c。<ol>
<li>将上清液转移到新的15毫升猎鹰管。</li>
	</ol>
</li>
	<li>用 blastR 稀释缓冲器将澄清的裂解液稀释为每150毫米板的最终体积为3毫升。最后的体积是基于预期的蛋白质产量 (表 2)。 注: 此步骤非常重要, 因为最终的缓冲组合将影响 IP 反应的严格。</li>
	<li>定量蛋白浓度 (2 节)。</li>
</ol>2. 蛋白质定量测定
注: 使用标准色度蛋白定量测定法测定蛋白质含量。在这里, 蛋白质定量是用精确的红色高级蛋白质测定。
<ol><li>在两个1毫升的试管中加入1毫升的精密红色高级蛋白质检测试剂。</li>
	<li>混合10µL blastR 裂解缓冲液和40µL blastR 稀释缓冲液在清洁1.5 毫升离心管冰。 注: 这将用于读取空白蛋白质样品。</li>
	<li>添加20µL 的裂解/稀释缓冲混合 (从步骤 2.2) 到第一试管和混合通过反转两到三倍。</li>
	<li>添加20µL 稀释细胞裂解 (从实验) 到第二试管, 混合如上所述。</li>
	<li>室温下孵育样品1分钟。</li>
	<li>空白分光光度计与溶解/稀释缓冲混合样品 (从步骤 2.3)。</li>
	<li>测量裂解样品的吸光度 (从步骤 2.4) 在 600 nm。</li>
	<li>确定裂解蛋白浓度如下; 样本读取 OD600 x 5 = 蛋白质浓度在毫克/毫升 注: OD 读数低于0.05 或以上0.5 是接近的线性范围的能力的蛋白质检测。对于读数 > 0.5, 样品可以稀释。对于读数 < 0.05, 更多的裂解液可以添加到精密的红色高级蛋白质检测试剂 (高达50µL)。请参见表 3以将分光光度计读数转换为毫克/毫升的裂解蛋白浓度。如果一个盘子里没有足够的蛋白质, 建议每个 IP 使用2或更多的盘子。在这种情况下, 板块将收获的系列, 转移原300µL 的裂解缓冲板之间。</li>
	<li>根据蛋白质浓度, 稀释样品与缓冲混合 (1 部分 blastR 裂解: 4 部分 blastR 稀释) 到一个理想的最终浓度 (通常1毫克/毫升)。</li>
	<li>对不会立即使用的示例进行快照冻结等分。<ol>
<li>在-70 ° c 贮存样品。</li>
		<li>进入3条。</li>
	</ol>
</li>
</ol>3. 沉淀 (IP) 检测
注意: 亲和珠浓度, 裂解浓度, 和孵化时间是推荐的准则, 可能是唯一的每一个目标蛋白和特定的 PTM 正在调查。
<ol><li>倒置的股票试剂管含有与之相关联的珠子, 有亲和力的珠子, 相扑2/3 亲和珠, 和 Ac 亲和珠几次, 以确保珠子是完全悬浮在管。</li>
	<li>对于每个 ip 检测, 分珠悬浮成单独的1.5 毫升离心管冰 (ip 管)。在这里, 添加20µL 泛素亲和珠, 30 µL 酪氨酸亲和珠, 40 µL sumo 2/3 亲和珠, 或50µL 乙酰化亲和珠到个别1.5 毫升离心管冰。 注意: 请参阅表 4 , 以了解每个关联珠的建议使用的磁珠悬浮量。</li>
	<li>反转库存试剂管含有泛的 IP 控制珠和 IgG 控制珠几次, 以确保珠子是完全悬浮在管。</li>
	<li>分控制珠每控制反应确定非特异性结合 (控制 IP 管)。在这里, 添加20µL 的布法罗控制珠, 30 µL 的控制珠, 40 µL 相扑2/3 控制珠, 或50µL 的 Ac 控制珠到个别1.5 毫升离心管冰。 注意: 请参阅表 4 , 建议为每种类型的亲和珠使用的磁珠悬浮液量。</li>
	<li>保存少量的裂解 (20 µL) 作为一个西方印迹输入裂解控制运行。添加5µL 的5x 样品缓冲, 煮沸在95° c 5 分钟。 注意: 有关缓冲区组合, 请参见材料表。</li>
	<li>将裂解液添加到每个 ip 管中, 并控制 ip 管。在这里, 1 毫克的裂解液使用每 ip 和控制反应, 导致总共8的 ip 反应。 注: 1.0 毫克的裂解液每一个化验建议作为出发点。所需的裂解液的数量会因修饰靶蛋白的丰度而异。</li>
	<li>在4° c 的涵管端旋转平台上孵育2小时的管子。在这里, 一个 ATR 管转子是使用在速度22。</li>
	<li>收集珠的离心在 3000-5000 x g 1 分钟, 在4° c。</li>
	<li>吸出尽可能多的上清, 而不干扰的珠子。</li>
	<li>洗涤珠在1毫升 blastR 洗涤缓冲 (抑制剂是不必要的在这个阶段) 为 5 min 在4° c 转动的平台。</li>
	<li>收集珠的离心在 3000-5000 x g 1 分钟, 在4° c。</li>
	<li>吸出尽可能多的上清, 而不干扰的珠子。</li>
	<li>重复洗涤步骤 (步骤 3.10-3.12) 两次。</li>
	<li>最后洗完后, 彻底清除上清液。最小的珠颗粒破坏是可以接受的 (高达5% 的损失)。用细孔蛋白加载尖端去除残留上清液。</li>
	<li>添加30µL 的珠洗脱缓冲, 并重的珠子轻轻拍打/弹的一侧管。在这个阶段不要使用吸管。 注意: 有关缓冲区组合, 请参见材料表。</li>
	<li>室温孵育5分钟。</li>
	<li>轻轻地将每个珠悬浮转移到一个1.5 毫升离心旋转柱, 已放置在一个1.5 毫升离心管。 注意: 建议将移液吸管尖端的末端剪断以进行更温和的转移。</li>
	<li>离心机在 9000-1万 x g 为1分钟在室温下收集 IP 样品。</li>
	<li>每样加2µL 2 基, 拌匀。 注意: 把盖子从旋转柱上拧开, 用它来盖收集管以作进一步的处理是很方便的。</li>
	<li>将样品放在95° c 的水浴中5分钟, 用离心法在 1万 x g 收集样品, 在 RT 处进行1分钟。</li>
	<li>如有必要, 将样品储存在-70 ° c, 停在这里, 或进行 SDS-页、转移和印迹分析 (4 节)。</li>
</ol>4. 免疫印迹分析: 鉴定蛋白质的兴趣
<ol><li>根据标准实验室协议32, 将十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 和转移到聚偏聚氟乙烯 (PVDF) 膜进行分离样品。</li>
	<li>使用一个主要的抗体来检测的 post-translationally 修改版本的蛋白质的兴趣。在这里, PD-L1 抗体被用来检测 post-translationally 修饰的 PD-L1。</li>
	<li>使用灵敏化学发光检测试剂进行检测。 注: 化学发光试剂应与 HRP 标记的二级抗体结合使用, 能检测主抗体。</li>
	<li>使用2毫升的化学发光试剂每 mini-gel-sized 转移膜 (约 8 x 7 厘米)。<ol>
<li>在孵育与适当的二次抗体 (60 分钟在 RT 推荐) 之后, 洗涤印迹 6 x 10 min 在30毫升的三缓冲盐水与 tween-20 (TBST)。 注意: 有关缓冲区组合, 请参见材料表。</li>
		<li>在使用前, 将1毫升的化学发光试剂 a 与1毫升的化学发光试剂 B 混合 (足够一 8 cm x 7 cm 膜)。</li>
		<li>使用 x 射线胶片或电荷耦合器件 (CCD) 摄像成像, 在 RT 中添加化学发光试剂到膜上, 在室温下孵育 1-5 分钟。 注: 在化学发光试剂中, 较短的潜伏期可能是高度丰富的蛋白质所必需的。</li>
	</ol>
</li>
</ol>

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朱合华和 a.1 是细胞骨架的雇员 Inc. k.m 是细胞骨架 Inc. 的创始人

用于确定目标蛋白是否由 PTM 修饰的初始方法可以使用目标蛋白特定的 ip 抗体进行, 其次是 PTM 抗体 (e. g, anti-acetyl 赖氨酸), 或使用 PTM 抗体为 ip,其次是免疫印迹与靶蛋白特异抗体30,31,33。虽然这两种方法的理论工作, 利用目标蛋白特异抗体有更多的潜在缺陷, 如抗体可能不是 IP 兼容, 或大 PTM 修改可能阻断抗体识别网站上的目标蛋白34 ,35。PTM 亲和珠的优点是抗体或结合域特别承认 PTM 的兴趣;因此, 对目标蛋白的修饰不应改变亲和珠的识别。以此为例, 用这里描述的技术进行了 PD-L1, 并观察到了内源性单和多聚物 (图 4)。Lim et al最近发布的一篇文章。利用体外技术来调查 PD-L1, 结果与图 436中显示的结果非常相似。有趣的是, 他们还执行了 PD-L1 抗体的 IP, 以丰富 PD-L1 从细胞裂解, 在那里表达和 MG-132 增加, 以提高信号。该模式是不健全的, 非常不同于体外的模式。细胞培养模型中内源性 PD-L1 的研究没有在 Lim et al中进行。报告以阐明其细胞培养与体外数据之间的差异。
调查一个蛋白质是否被 PTM 修改可能是挑战, 由于它的丰盈和瞬态的自然37,38, 并且经常需要通过 IP 丰富。PTMs 的有效 IP 需要优化几个关键步骤和试剂, 如裂解缓冲剂和亲和试剂。当调查目标蛋白的多个 PTMs 时, 所需的优化可能会增加。利用 blastR 裂解系统是该协议的关键一步, 因为它保持了强健的 IP 能力, 同时在一个系统中能够对 PTM、相扑2/3、布法罗和 Ac PTMs 进行检测。此技术优化了确定特定目标蛋白是否由这四 PTMs 进行修改所需的时间和资源, 并可能提供更好的 PTM 串扰的图片, 相对于使用多个裂解系统进行的 PTM 结果比较。对 blastR 裂解体系与替代 PTMs (如糖基化) 的相容性进行了研究;然而, 对所有类型的 PTMs 都没有进行过详尽的检查。
丰富的基因组 DNA 可以通过色度或 nanodrop 的方法干扰蛋白质的测量, 影响 SDS 丙烯酰胺凝胶中蛋白质的迁移, 并在 IP 分析过程中防止蛋白质和亲和基质相互作用。这里描述的方法利用一个专门的过滤器有效地去除基因组 DNA 污染, 这是该协议的另一个关键步骤。为了突出这一点, 在 blastR 过滤器处理前后进行了粘度测试, 结果显示, 从高粘度到水的粘度降低 (数据没有显示)。在图 3中的结果支持了这种粘度的变化, 显示几乎所有的基因组 DNA 都被移除。重要的是, dna 不是用这种方法剪切的, 因为任何剪切的 dna 都不会被过滤器捕获 (数据没有显示)。这个工具优于传统的方法, 如超声或注射器-dna 剪切, 因为它不需要专门的设备, 是高度重现性, 并删除 dna, 而不是剪切它。此外, 它不会降低在裂解液中的蛋白质, 这可能会发生使用传统的方法29。与其他方法相比, 使用过滤器需要 5-30 秒的时间, 而 dna 的有效击穿可能需要几分钟 (即, 注射器剪切), 并且可能导致样品的异质性, 因为 dna 污染物残留在裂解物中。对裂解物和 post-blastr 的筛选进行了广泛的分析, 马斯亮、靶向性的西方、总的和靶向特异的 PTM 分析均未观察到蛋白质剖面的显著差异;因此, 筛选出的基因组 DNA 可能不会影响蛋白质的完整性。最终, 这个过滤系统是有益的任何西方或 IP 应用的基因组 DNA 存在, 并可能影响解释的蛋白质分析。
重要的是要注意, 有潜在的假阴性检测利用这种技术, 这可能是由于亲和力珠饱和度, 结合现场干扰, 或 PTM 掩蔽。例如, 某一特定靶蛋白可以通过 Ac 在极低的水平上进行修改;因此, 它可能不会被孤立的泛乙酰赖氨酸亲和珠已饱和的更丰富的交流修饰目标蛋白。目前正在进行研究, 以评估该协议中所使用的亲和试剂的检测限度, 但最近的一份出版物表明了非常可靠的检测限制。数据显示, 这项技术可以确定每单元的17乙酰目标蛋白分子的31。尽管如此, 特定的情况, 如细胞类型特异性, 瞬态 PTMs 响应特定的刺激, 或掩蔽 PTMs 由于蛋白质相互作用可能导致假阴性结果。这些是这种技术的潜在缺陷, 以及大多数 PTM 的 IP 方法。因此, 建议使用多种方法来确认结果。
如糖基化和磷酸化的修改已经显示出竞争类似的氨基酸39,40, 和其他 PTMs 像泛和磷酸酯已被证明工作顺序, 以调节蛋白质的函数17,41。最近关于病理蛋白 tau 的研究突出了 PTM 串扰的重要性, 其中 tau 超磷酸化和 tau sumo 增强了彼此的42。此外, 该组显示, sumo 的头防止了多聚和随后的 tau 退化, 可能导致聚集。这只是许多管理 PTM 串扰的例子之一, 这项技术的效用将有助于阐明 PTMs 及其串扰在调节健康和疾病关键蛋白中的重要性。

翻译后的修改 (PTMs) 是对蛋白质的高度调控的改变, 藉以特定的方式增加或去除修改。PTMs 通常是动态的, 瞬变的变化, 极大地改变了蛋白质的结构, 与伙伴蛋白质的相互作用, 和空间定位, 并最终使蛋白质执行不同的功能1,2,3,4. PTMs 是如此丰富, 他们增加了独特的蛋白质形式 (或 proteoforms) 从大约3万基因产品的数量, 以超过 100万 proteoforms5,6。识别 PTMs 并定义其对靶蛋白的影响对于了解蛋白质的生理和病理功能是至关重要的7,8,9,10, 11、12、13、14。蛋白、p53 和表皮生长因子受体 (EGFR) 等选择蛋白的工作已经阐明了 PTMs15、16、17的调节作用。这些研究揭示了这样一个事实, 即这些蛋白质的调节发生在多个 PTMs, 在许多情况下, 这些修改的工作, 以促进一个特定的功能。新的研究表明, 合作和负 PTM 串扰可以发生在几个不同的蛋白质18,19,20,2122, 23,24,25。然而, 大多数蛋白质的 PTM 剖面是建立在几个研究, 使用了不同的模型和独特的条件。有一个优化系统, 以调查多个 PTMs 在一个系统将是非常有益的洞察潜在的 PTM 串扰的目标蛋白。
一个挑战与调查 PTMs 在一个单一的系统是, 特定的 PTMs 被调查使用不同的裂解缓冲。例如, 通常用于磷酸化或泛调查的缓冲 (如帕或 NP-40) 的使用可能不足以研究不稳定的 PTM, 如小泛素样修饰剂 (相扑) ylation26。此外, 变性缓冲区不足以游离蛋白质的相互作用, 并可能导致假阳性 PTM 识别27。调查 PTMs 的变性裂解缓冲可能是首选的, 因为它是明显更好地隔离蛋白质从所有细胞室28, 将离解大多数蛋白质的相互作用, 并将抑制蛋白酶, 改变 PTM 状态, 如deSUMOylases 26;然而, 变性缓冲器可能损害特定亲和试剂 (如泛素结合基于域的工具27) 的完整性。开发一种类似变性的裂解系统来研究亲和试剂相容系统中蛋白质的多重 PTMs, 对于 PTM 串扰的调查是非常有益的。
虽然变性缓冲器的主要优势超过变性缓冲区的调查 PTMs, 他们很少使用, 因为它们的重大缺陷, 如与传统的蛋白质检测不相容, 重要的基因组脱氧核糖核酸 (脱氧核糖核酸) 污秽和中断对沉淀 (IP) 试剂29。这些缺点可能导致延长准备时间和潜在的损害目标蛋白时, 剪切基因组 DNA。用于剪切 DNA 的两种常用方法包括注射器裂解和超声29。这两种方法都需要广泛的优化, 而且往往缺乏精确的重现性。除去基因组 DNA 的替代方法包括增加 DNAses;但是, 这可能需要额外的优化和缓冲区组成的变化。最终, 一个简单和可重现的方法, 以消除基因组 DNA 污染将有利于在使用变性裂解缓冲剂的 PTM 调查。
这项技术的目的是建立一个系统, 孤立和调查泛 (布法罗), 酪氨酸磷酸化, sumo 2/3 (相扑 2/3), 和乙酰 (Ac) PTMs 在一个单一的系统, 以更好地研究 PTM 串扰的目标蛋白。利用该 PTM 检测系统, 对单个系统中几种靶蛋白的内源 PTM 剖面进行了快速研究。对潜在的串扰的新洞察被辨认了30,31。总的来说, 这里描述的技术改进了现有的方法来调查 PTMs 和 PTM 串扰在三种不同的方式: 1) 一个独特的变性缓冲被开发, 分离蛋白质从所有细胞室, 同时仍然是兼容PTM 亲和试剂, 2) 一个独特的过滤系统的发展, 迅速消除基因组 DNA 污染的变性裂解具有高重现性, 不需要专门的设备, 和 3) 强大的亲和珠, 裂解系统, 抑制试剂是相容的;因此, 简化了这四 PTMs 的分离, 为目标蛋白最大化 PTM 富集, 并有效地检查潜在的串扰。

<table><tbody><tr><td>&lt;strong&gt;Cell lysis/genomic DNA removal and analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1x phosphate buffered saline (PBS)</td><td>common buffer</td><td></td><td>Recipe: NaCl, KCl, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>BlastR lysis buffer</td><td>Cytoskeleton Inc.</td><td>BLST01</td><td>Proprietary mixture of detergents, salts, and other buffer additives</td></tr><tr><td>Radioimmunoprecipitation assay buffer (RIPA)</td><td>common buffer</td><td></td><td>Recipe: Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Igepal</td></tr><tr><td>Mammalian protein extraction reagent (mPER)</td><td>Thermofisher</td><td>78503</td><td>Proprietary mixture of detergents, salts, and other buffer additives</td></tr><tr><td>Immunoprecipitation (IP) Lysis</td><td>Pierce</td><td>87787</td><td>Proprietary mixture of detergents, salts, and other buffer additives</td></tr><tr><td>1% sodium dodecyl sulfate (SDS) denaturing buffer</td><td>common buffer</td><td></td><td>Recipe: PBS, SDS, EDTA, EGTA</td></tr><tr><td>Laemmli</td><td>common buffer</td><td></td><td>Recipe: Tris-HCl, SDS, glycerol, bromophenol blue</td></tr><tr><td>BlastR dilution buffer</td><td>Cytoskeleton Inc.</td><td>BDB01</td><td>Proprietary mixture of detergents, salts, and other buffer additives</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Cytoskeleton Inc.</td><td>PIC02</td><td></td></tr><tr><td>N-Ethylmaleimide (NEM) &amp;amp; N,N,N&amp;prime;,N&amp;prime;-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)</td><td>Cytoskeleton Inc.</td><td>NEM09BB</td><td></td></tr><tr><td>Trichostatin A (TSA) &amp;amp; Nicotinamide</td><td>Cytoskeleton Inc.</td><td>TSA01</td><td></td></tr><tr><td>Activated Sodium Orthovanadate (Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4)&lt;/sub&gt;</td><td>Cytoskeleton Inc.</td><td>PYI01</td><td></td></tr><tr><td>cell scrapers</td><td>Costar</td><td>3008</td><td></td></tr><tr><td>BlastR Filter</td><td>Cytoskeleton Inc.</td><td>BLR02</td><td></td></tr><tr><td>BD Insulin syringe needle</td><td>BD</td><td>08290-3284-11</td><td></td></tr><tr><td>sonicator (Branson Sonifier)</td><td>Branson</td><td>Sonifier 450</td><td></td></tr><tr><td>Precision Red Advanced Protein Reagent</td><td>Cytoskeleton Inc.</td><td>ADV02</td><td></td></tr><tr><td>1 mL cuvettes</td><td>Fisher</td><td>14955127</td><td></td></tr><tr><td>Spectrophotometer (Genesys20)</td><td>Thermofisher</td><td>4001</td><td>Other spectrophotomers can be used to measure protein concentration</td></tr><tr><td>Agarose</td><td>Fisher</td><td>BP1356-500</td><td></td></tr><tr><td>1kb Deoxyribonucleic acid (DNA) ladder</td><td>NEB</td><td>N3232L</td><td></td></tr><tr><td>DNA gel box</td><td>Thermofisher</td><td>7312 B1A</td><td></td></tr><tr><td>Tris/Borate/EDTA (TBE) buffer</td><td>common buffer</td><td></td><td>Recipe: Tris-HCl, Boric Acid, EDTA</td></tr><tr><td>&lt;strong&gt;PTM Immunoprecipitation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphotyrosine beads</td><td>Cytoskeleton Inc.</td><td>APY03-beads</td><td></td></tr><tr><td>Ubiquitination beads</td><td>Cytoskeleton Inc.</td><td>UBA01-beads</td><td></td></tr><tr><td>SUMOylation 2/3 beads</td><td>Cytoskeleton Inc.</td><td>ASM24-beads</td><td></td></tr><tr><td>Acetylation beads</td><td>Cytoskeleton Inc.</td><td>AAC04-beads</td><td></td></tr><tr><td>Phosphotyrosine control beads</td><td>Cytoskeleton Inc.</td><td>CIG01-beads</td><td></td></tr><tr><td>Ubiquitination control beads</td><td>Cytoskeleton Inc.</td><td>CUB02-beads</td><td></td></tr><tr><td>SUMOylation 2/3 control beads</td><td>Cytoskeleton Inc.</td><td>CIG01-beads</td><td></td></tr><tr><td>Acetylation control beads</td><td>Cytoskeleton Inc.</td><td>CIG02-beads</td><td></td></tr><tr><td>BlastR wash buffer</td><td>Cytoskeleton Inc.</td><td>BWB02</td><td>Proprietary mixture of detergents, salts, and other buffer additives</td></tr><tr><td>Bead elution buffer</td><td>Cytoskeleton Inc.</td><td>BEB01</td><td>Proprietary mixture of detergents, salts, and other buffer additives</td></tr><tr><td>microcentrifuge</td><td>Eppendorf</td><td>5417</td><td>Other microcentrifuges can be used&amp;nbsp;</td></tr><tr><td>tube rotator</td><td>ATR</td><td>RKVSD</td><td>Other end-over-end tube rotators can be used&amp;nbsp;</td></tr><tr><td>protein loading tips</td><td>VWR</td><td>37001-150</td><td></td></tr><tr><td>spin columns</td><td>Cytoskeleton Inc.</td><td>SPN22</td><td></td></tr><tr><td>heat block 95 &amp;deg;C</td><td>Benchmark</td><td>BSH1002</td><td></td></tr><tr><td>&lt;strong&gt;SDS-PAGE/Transfer/Western analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5x sample buffer</td><td>common buffer</td><td></td><td>recipe: Bromophenol blue, dithiothreitol (DTT), Glycerol, SDS, Tris-HCl</td></tr><tr><td>novex wedge well 4-20% Tris-gly gels</td><td>Invitrogen</td><td>XP04200BOX</td><td>The large wedgewells are excellent for loading large volumes</td></tr><tr><td>1x Running buffer</td><td>common buffer</td><td></td><td>recipe: Tris-HCl , Glycine, SDS</td></tr><tr><td>seeblue protein ladder</td><td>Life Technologies</td><td>LC5625</td><td></td></tr><tr><td>electrophoresis cell</td><td>Invitrogen</td><td>Xcell surelock electrophoresis cell</td><td>Other electrophoresis cells can be used, however these are compatible the novex wedgewell gels&amp;nbsp;</td></tr><tr><td>power supply</td><td>Biorad</td><td>PowerPac HC</td><td>Other power supplies can be used&amp;nbsp;</td></tr><tr><td>1x Transfer buffer</td><td>common buffer</td><td></td><td>recipe: Tris-HCl, glycine, methanol</td></tr><tr><td>Transfer cell</td><td>Biorad</td><td>mini trans-blot cell</td><td>Other transfer cells can be used&amp;nbsp;</td></tr><tr><td>Immobilon- P membranes (PVDF)</td><td>millipore</td><td>IPVH 304F0</td><td></td></tr><tr><td>non-fat milk</td><td>thrive</td><td>813503015095</td><td></td></tr><tr><td>Tris buffered saline with tween (TBST)</td><td>common buffer</td><td></td><td>recipe: Tris-HCl, NaCl, Tween 20</td></tr><tr><td>Chemiluminescent Detection Reagent</td><td>Cytoskeleton Inc.</td><td>CLRA/CLRB</td><td></td></tr><tr><td>&lt;strong&gt;Cell growth and treatment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s medium&amp;nbsp;</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ATLAS</td><td>&amp;nbsp;F-0500-A&amp;nbsp;</td><td></td></tr><tr><td>Epidermal growth factor</td><td>Cytoskeleton Inc.</td><td>CN02-A</td><td></td></tr><tr><td>150 mm cell culture plates</td><td>Corning</td><td>430599</td><td></td></tr></tbody></table>

utilizing a comprehensive immunoprecipitation enrichment system to identify an endogenous post-translational modification profile for target proteins

现在欢迎的是, 修饰修饰 (PTMs) 在调节蛋白质的结构和功能方面起着不可或缺的作用, 这对于特定蛋白质的生理和病理作用是必不可少的。在研究目标蛋白的 PTM 状态时, PTMs 的富集常常是必要的, 因为 PTMs 往往是短暂的, 而且丰度相对较低。当对目标蛋白的 PTM, 如缓冲不相容, 靶蛋白抗体不兼容, 丢失 PTM 信号等, 会遇到很多陷阱。在研究多种 PTMs 如乙酰化、泛、sumo 2/3 和酪氨酸磷酸化为特定靶蛋白时, 难度会放大。研究这些 PTMs 的组合可能是必要的, 因为 PTMs 之间的串扰是普遍和关键的蛋白质调节。通常, 这些 PTMs 在不同的裂解缓冲剂和独特的抑制剂组合物中进行研究。为了简化这一过程, 开发了一种独特的变性裂解系统, 有效地分离和保存这四 PTMs;因此, 在单个裂解系统中启用潜在串扰的调查。一个独特的过滤系统的设计, 以消除污染的基因组 DNA 的裂解, 这是一个问题 by-product 的变性缓冲区。针对四 PTMs 中每一个的鲁棒亲和矩阵与缓冲系统协同发展, 以最大限度地丰富和检测这四 PTMs 的内生状态。这一全面的 PTM 检测工具集简化了获取关键信息的过程, 即蛋白质是否由其中一个或多个 PTMs 修改。

这些工具的能力, 以调查 PTM 串扰通过有效地检测这 4 PTMs 部分依赖于独特的裂解缓冲系统。blastR 变性缓冲有效地将蛋白质从所有细胞室中分离出来, 类似于其他变性缓冲器, 从而确保了完整的蛋白质配置 (图 2A)。此外, 它保留了高度不稳定的 PTM 信号, 如相扑 2/3, 这是迅速减少在变性缓冲像 radioimmunoprecipitation 检测 (帕) (图 2B)。重要的是, 当适当稀释时, 它不会像其他变性缓冲器那样影响亲和性试剂的完整性 (例如, Laemmli 缓冲区)。
在处理变性缓冲器时, 一个重要的障碍是有效去除基因组 DNA 污染的能力。传统的降低粘度的方法是使用注射器针或 sonicating 样品来剪切 DNA。图 3A显示了 BlastR 过滤器、注射器针或超声治疗后 A431 细胞的基因组 DNA 污染。有几乎完全去除基因组 DNA 使用 BlastR 过滤器, 这不是情况下使用常规注射器针或超声。基因组 DNA 污染通过 SDS-丙烯酰胺凝胶对蛋白质的迁移有显著影响;然而, 与 BlastR 过滤器的治疗去除基因组 DNA, 导致适当的蛋白质迁移 (图 3B)。基因组 DNA 污染引起的迁移改变对西方印迹的解释有显著影响;例如, 在未筛选的裂解液中看到的表皮生长的 EGFR 模式可能不准确地解释为相对于筛选样本的增加表达式 (图 3C)。
通过利用这种优化的 PTM 浓缩和检测系统, 可以快速确定一个或多个 PTMs 的目标蛋白是否被修改. PD-L1 PTM 剖面的研究是最近使用该技术进行的, 结果表明 PD-L1 是化, 乙酰, 酪氨酸磷酸化反应 EGF (图 4)。重要的是, 这些数据报告内源性 PD-L1 PTM 的变化, 这代表了一个小百分比的总 PD-L1 确定。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56912/56912fig1.jpg"> 图 1: 使用 BlastR 过滤器从细胞裂解液中过滤基因组 DNA.(A) BlastR 筛选器的图像。(B)裂解液加载到放置在15毫升收集管中的过滤器中。(C)将柱塞放入注射器中, 并通过压缩将裂解液通过过滤器。(D)收集裂解液, 包括通过完全压缩进行的任何气泡。(E)筛选出的裂解液。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56912/56912fig2.jpg"> 图 2: BlastR 裂解缓冲液与替代裂解缓冲器的比较.图 2A改编自堀et al.2017. Biosci。代表。31(A) A431 细胞裂解 BlastR、帕、mPER、IP 裂解、变性 (1% SDS) 和 Laemmli 裂解缓冲液。所有变性裂解都使用 BlastR 过滤器去除了基因组 DNA。从细胞膜、细胞质、线粒体和细胞核标记中分离出蛋白质, 用抗体对各自的间隔标记蛋白进行测定。(B) A431 细胞与 BlastR、帕和 1% SDS 变性缓冲裂解。裂解是 immunoprecipitated 与相扑2/3 或控制 IgG 亲和珠。用 2/3-辣根过氧化物酶 (HRP) 抗体对样品进行了分离, 并用西印迹进行了分析。N≥3独立实验的典型印迹。<a href="//cloudflare.jove.com/files/ftp_upload/56912/56912fig2large.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/56912/56912fig3.jpg"> 图 3: BlastR 裂解过滤器能有效去除基因组 DNA.(a) A431 细胞与变性裂解缓冲裂解。在5、10、20或30秒内, 用 BlastR 过滤器、注射器针或超声去除或剪切基因组 DNA。用溴溴化琼脂糖凝胶电泳法对2% 种裂解物进行了分析。(B)从带有变性缓冲区的 A431 细胞裂解的裂解液未经过滤或用 BlastR 过滤器过滤。样品与 SDS 页分离, 使用马斯亮染色进行可视化。(C) B 中的重复样本由 SDS 页分隔, 转移到 PVDF, 并使用 egfr 抗体检测 egfr 蛋白。给出了3独立实验的典型印迹。<a href="//cloudflare.jove.com/files/ftp_upload/56912/56912fig3large.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/56912/56912fig4.jpg"> 图 4: EGF 诱导的 PD-L1 修饰修饰的检测.图改编自堀et al.2017.肿瘤30(A)。血清限制性 A431 细胞要么刺激 (UT), 要么刺激与 EGF 一小时前裂解 BlastR 裂解缓冲液。世界劳联被分析为 PD-L1 水平 (车道 12)。泛素结合珠 (UBA01) 被用于 IP 化蛋白质 (车道 34)。酪氨酸结合珠 (APY03) 被用于 IP 酪氨酸磷酸化蛋白 (车道 56)。相扑2/3 结合珠 (ASM24) 被用于 IP SUMOylated 2/3 蛋白质 (车道 78)。乙酰赖氨酸结合珠 (AAC01) 被用于 IP 乙酰蛋白质 (车道 910)。IgG 结合控制珠被用于 IP 非特异结合蛋白 (车道 1112)。采用 SDS 页分离样品, 用 PD-L1 抗体进行印迹分析。给出了3独立实验的代表性印迹。白星号用于突出 PD-L1 和 Ac 蛋白带。<a href="//cloudflare.jove.com/files/ftp_upload/56912/56912fig4large.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 colspan="4">BlastR 裂解缓冲液的计算</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>1.0 毫升</td>
			<td>2.0 毫升</td>
			<td>5.0 毫升</td>
			<td>10.0 毫升</td>
		</tr>
<tr>
<td>BlastR 裂解缓冲液</td>
			<td>965µL</td>
			<td>1930µL</td>
			<td>4.825 毫升</td>
			<td>9.650 毫升</td>
		</tr>
<tr>
<td>酪氨酸磷酸酶抑制剂</td>
			<td>5µL</td>
			<td>10µL</td>
			<td>25µL</td>
			<td>50µL</td>
		</tr>
<tr>
<td>de-ubiquitinase/de-sumoylase 抑制剂</td>
			<td>10µL</td>
			<td>20µL</td>
			<td>50µL</td>
			<td>100µL</td>
		</tr>
<tr>
<td>HDAC 抑制剂</td>
			<td>10µL</td>
			<td>20µL</td>
			<td>50µL</td>
			<td>100µL</td>
		</tr>
<tr>
<td>蛋白酶抑制剂鸡尾酒</td>
			<td>10µL</td>
			<td>20µL</td>
			<td>50µL</td>
			<td>100µL</td>
		</tr>
<tr>
<td colspan="4">BlastR 稀释缓冲器的计算</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>4.0 毫升</td>
			<td>8.0 毫升</td>
			<td>20.0 毫升</td>
			<td>40.0 毫升</td>
		</tr>
<tr>
<td>BlastR 裂解缓冲液</td>
			<td>3.86 毫升</td>
			<td>7.72 毫升</td>
			<td>19.3 毫升</td>
			<td>38.6 毫升</td>
		</tr>
<tr>
<td>酪氨酸磷酸酶抑制剂</td>
			<td>20µL</td>
			<td>40µL</td>
			<td>100µL</td>
			<td>200µL</td>
		</tr>
<tr>
<td>de-ubiquitinase/de-sumoylase 抑制剂</td>
			<td>40µL</td>
			<td>80µL</td>
			<td>200µL</td>
			<td>400µL</td>
		</tr>
<tr>
<td>HDAC 抑制剂</td>
			<td>40µL</td>
			<td>80µL</td>
			<td>200µL</td>
			<td>400µL</td>
		</tr>
<tr>
<td>蛋白酶抑制剂鸡尾酒</td>
			<td>40µL</td>
			<td>80µL</td>
			<td>200µL</td>
			<td>400µL</td>
		</tr>
</tbody></table>表 1:溶解和稀释缓冲准备图.在为细胞裂解准备缓冲液时, 提供抑制剂浓度以增加给定的溶解和稀释缓冲体积的图表。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>板蛋白含量</td>
			<td>推荐 BlastR 裂解缓冲卷</td>
			<td>推荐 BlastR 稀释缓冲容积</td>
		</tr>
<tr>
<td>< 1 毫克</td>
			<td>将蛋白质与多个板块结合</td>
			<td>使1.5 毫升最终体积</td>
		</tr>
<tr>
<td>1-2 毫克</td>
			<td>300µL</td>
			<td>使1.5 毫升最终体积</td>
		</tr>
<tr>
<td>2-4 毫克</td>
			<td>600µL</td>
			<td>使3毫升最终体积</td>
		</tr>
<tr>
<td>4-6 毫克</td>
			<td>900µL</td>
			<td>使4.5 毫升最终体积</td>
		</tr>
</tbody></table>表 2:BlastR 溶解/稀释缓冲图在获得裂解液时, 提供建议的溶解和稀释缓冲容积的图表。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>添加到1毫升的精密红蛋白测定试剂 (µL) 的细胞裂解量</td>
			<td>用于示例读取 OD 的乘数600</td>
		</tr>
<tr>
<td>10</td>
			<td>10</td>
		</tr>
<tr>
<td>20</td>
			<td>5</td>
		</tr>
<tr>
<td>30</td>
			<td>3。3</td>
		</tr>
<tr>
<td>40</td>
			<td>2。5</td>
		</tr>
<tr>
<td>50</td>
			<td>2</td>
		</tr>
</tbody></table>表 3:乘法器将分光光度计读数转换为毫克/毫升的裂解产物.图表提供转换号码, 以计算蛋白质浓度的助手。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>亲和珠</td>
			<td>珠浆/IP 容积 (毫升)</td>
		</tr>
<tr>
<td>泛</td>
			<td>20</td>
		</tr>
<tr>
<td>酪氨酸</td>
			<td>30</td>
		</tr>
<tr>
<td>sumo 2/3</td>
			<td>40</td>
		</tr>
<tr>
<td>乙酰</td>
			<td>50</td>
		</tr>
<tr>
<td>控制珠</td>
			<td>珠浆/IP 容积 (毫升)</td>
		</tr>
<tr>
<td>泛控制珠</td>
			<td>20</td>
		</tr>
<tr>
<td>Phosphotyrsoine 控制珠</td>
			<td>30</td>
		</tr>
<tr>
<td>sumo 2/3 控制珠</td>
			<td>40</td>
		</tr>
<tr>
<td>乙酰化控制珠</td>
			<td>50</td>
		</tr>
</tbody></table>表 4:建议的珠子/IP 图卷.图表提供建议数量的亲和力珠子使用每个 IP 反应。

Watch this Scientific Journal Video about Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins at JoVE.

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

研究多, 内源性修饰修改的目标蛋白可能是极具挑战性的。这里描述的技术利用一个优化的裂解缓冲和过滤系统开发了特定的 PTM 靶向, 亲和矩阵, 以检测乙酰化, 泛, sumo 2/3 和酪氨酸磷酸酯的修改为目标蛋白质使用单一的, 流线型的系统。

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

utilizing-comprehensive-immunoprecipitation-enrichment-system-to

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.2K 次观看.  Cytoskeleton Inc.. 研究多, 内源性修饰修改的目标蛋白可能是极具挑战性的。这里描述的技术利用一个优化的裂解缓冲和过滤系统开发了特定的 PTM 靶向, 亲和矩阵, 以检测乙酰化, 泛, sumo 2/3 和酪氨酸磷酸酯的修改为目标蛋白质使用单一的, 流线型的系统。

视频: Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

科研

生物化学

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

生物学

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

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

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

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

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

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological matrices is often necessary due to their low stoichiometry in comparison to non-modified analytes. Most enrichment workflows of PTMs on peptides in bottom-up proteomics workflows, where proteins are enzymatically digested before the resulting peptides are analyzed, only enrich one type of modification. It is the entire complement of PTMs, however, that leads to biological functions, and enrichment of a single type of PTM may miss such crosstalk of PTMs. PTM crosstalk has been observed between protein glycosylation and phosphorylation, the two most common PTMs in human proteins and also the two most studied PTMs using mass spectrometry workflows. Using the simultaneous enrichment strategy described herein, both PTMs are enriched from post-mortem human pancreatic tissue, a complex biological matrix. Dual-functional Ti(IV)-immobilized metal affinity chromatography is used to separate various forms of glycosylation and phosphorylation simultaneously in multiple fractions in a convenient spin tip-based method, allowing downstream analyses of potential PTM crosstalk interactions. This enrichment workflow for glyco- and phosphopeptides can be applied to various sample types to achieve deep profiling of multiple PTMs and identify potential target molecules for future studies.

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

一种同时分析人胰腺组织中N-糖肽和磷酸肽的自旋尖端富集策略

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

量化和站点本地化两个翻译后修改的同步亲和力丰富

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...