Name: detection of protein s-acylation using acyl-resin assisted capture
Uploaded: 2020-04-10T15:00:00
Description: Watch this Scientific Journal Video about Detection of Protein S-Acylation using Acyl-Resin Assisted Capture at JoVE.com

这项工作得到了国家卫生研究院提供的5R01GM115446和1R01GM130840的支持。

根据NIH准则，该协议中使用的小鼠被安乐死。休斯敦得克萨斯大学健康科学中心的动物福利委员会批准了所有动物工作。
1. 细胞水球的制备
<ol><li>准备如表 1中所述的集塞缓冲区。到 10 mL 的 PBS 中，加入 0.1 克 n-多代乙酯 +D-麦芽糖洗涤剂 （DDM）并旋转以溶解。加入100 μL磷酸酶抑制剂鸡尾酒2、ML211（10μM）、PMSF（10 mM）和蛋白酶抑制剂鸡尾酒（1倍），并在使用前...

<ol>
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在这里，我们成功地利用acyl-RAC测定检测培养的人类细胞和从小鼠组织中提取的原发细胞中选定蛋白质的S-化。该方法简单、灵敏，使用标准生物化学技术，在设备要求极小的情况下，易于执行。该方法已被证明能够成功地识别新的S-囊肿蛋白，如蛋白质转移系统（Sec61b）、核糖蛋白S11（Rps11）和微体谷胱甘肽-S-转移酶3（MGST3）22。22acyl-RAC的敏感性和适应性使其适合在各种?...

S-脱硫是一种可逆的翻译后修饰，涉及通过拉迪尔硫酯键1在靶蛋白上加入一个脂肪酸酶链到目标蛋白上的内半胱氨酸残留物。它最初被报道为用棕榈酸，一种饱和脂肪酸2的蛋白质的修饰，因此这种修饰通常被称为S-棕榈酸。除了棕榈酸盐，蛋白质可以通过各种更长和较短的饱和（硫酸和凝结酸）、单不饱和（油酸盐）和多不饱和（烷基酮和异氨酯酸）脂肪酸33、4、5、6、74,5,6,7可逆性地改变。在真核细胞中，S-acycye是由一种称为DHHC蛋白酶的酶家族催化的，半胱氨酸脱硫酶的反向反应被蛋白硫酯酶催化，其中大多数仍然保持神秘的8。
硫化物键的可浸性使这种脂质修饰可逆，使其能够动态调节蛋白质簇簇、血浆膜定位、细胞内贩运、蛋白质-蛋白质相互作用和蛋白质稳定性9,9、10。因此，S-囊肿与亨廷顿舞蹈症、阿尔茨海默病和几种癌症（前列腺、胃癌、膀胱癌、肺癌、结肠直肠癌）等疾病有关，因此需要开发可靠的方法来检测这种转化后蛋白修饰11。
代谢标记与放射性（[3H]，[14C]或[125I]）棕榈酸是最早开发的方法之一，以测定蛋白质S-囊14化12，13，14。12,13,14然而，基于放射性标签的方法存在健康问题，不是很敏感，耗时，只检测高丰富的蛋白质15的脂质化。一种更快、非放射性的放射性替代无线电标签是代谢标签与生物正交脂肪酸探针，这是例行用于检测蛋白质S-囊肿16的动态。在这种方法中，含有化学报告器（烷基或阿齐德组）的脂肪酸通过蛋白质转质酶被结合到S-青化蛋白中。Azide-alkyne Huisgen环增反应（点击化学）然后可用于将一个功能化组，如氟磷或生物素，连接到一体化脂肪酸，允许检测S-囊青蛋白17，18，19。17,18,19
Acyl-生物锡交换（ABE）是广泛采用的生物化学方法之一，用于捕获和鉴定S-囊肿蛋白，绕过代谢标记的一些缺点，如不适合组织样本15。该方法可用于分析各种生物样品的S-化，包括组织和冷冻细胞样本20、21。20,该方法基于中性羟基胺对酸基组和半胱氨酸残留物之间的三酯键的选择性裂解。然后，用三醇反应生物组子捕获。生成的生物微化蛋白然后使用链球菌加罗丝进行亲和力纯化，并通过免疫凝血进行分析。
一种称为乙基树脂辅助捕获（Acyl-RAC）的替代方法后来被引入，用游脱反应树脂22，23,23取代生物微化步骤，直接结合游自由半胱氨酸。与ABE相比，这种方法的步骤较少，同样可用于检测各种样品1的蛋白质S-青化。
Acyl-RAC 由 4 个主要步骤（图 1） 组成 。 1. 阻止自由三醇组; 2. 选择性地裂解半胱氨酸-酸酯结合与中性羟基胺（HAM），以暴露半胱氨酸硫醇群; 3. 用三醇反应性树脂捕获脂质化半胱氨酸; 4. 洗脱后选择性富集S-青化蛋白，减少缓冲。
然后，通过免疫凝血或进行基于质谱（MS）的质谱（MS）分析捕获的蛋白质，以评估不同种类和组织22、24、25的S-cycytttome。22,24,25通过对捕获的蛋白质的胰蛋白消化和LC-MS/MS22分析产生的肽，也可以识别个别S-青化位点。在这里，我们演示了如何使用 acyl-RAC 同时检测细胞系和组织样本中多个蛋白质的 S-acy 化。

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			<td height="21" style="height:21px;width:216px;">cOmplete Protease Inhibitor Cocktail tablets</td>
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			<td height="21" style="height:21px;">Eppendorf Centrifuge 5424</td>
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			<td height="21" style="height:21px;">Hydroxylamine (HAM)</td>
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			<td height="21" style="height:21px;">Methyl methanethiosulfonate (MMTS)</td>
			<td>Sigma</td>
			<td align="right" style="width:119px;">64306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini tube rotator</td>
			<td>LabForce</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ML211</td>
			<td>Cayman</td>
			<td align="right" style="width:119px;">17630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-Therm Cool-Heat-Shake</td>
			<td>Benchmark Scientific</td>
			<td style="width:119px;">H5000-HC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Dodecyl &#946;-D-maltoside (DDM)</td>
			<td>Sigma</td>
			<td style="width:119px;">D641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphatase Inhibitor Cocktail 2</td>
			<td>Sigma</td>
			<td style="width:119px;">P5726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiopropyl-Sepharose 6B (TS)</td>
			<td>Sigma</td>
			<td style="width:119px;">T8387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonics Quantrex Sonicator</td>
			<td>L &#38; R</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of protein s-acylation using acyl-resin assisted capture

蛋白质S-囊化，也称为S-Palmiyyyy化，是通过小链脂肪酸，通过小链脂肪酸对半胱氨酸残留物进行可逆的转化后修饰。S-acy化正在成为一种广泛的调节机制，它可以调节蛋白质生物活性的所有方面，从复杂的形成到蛋白质的贩运和蛋白质的稳定性。最近对蛋白质S-脱化生物功能的理解取得了进展，这主要是因为新型生化工具的开发，使各种生物样品中蛋白质S-脱化能够进行可靠和灵敏的检测。在这里，我们描述了乙基树脂辅助捕获（Acyl-RAC），这是一种最近开发的方法，基于通过硫醇反应性Sepharose珠选择性捕获内质S-囊肿蛋白。与现有方法相比，Acyl-RAC 需要较少的步骤，当与质谱法相结合以识别新型 S-循环目标时，可以产生更可靠的结果。该技术的一个主要限制是缺乏通过同一硫酯键区分附着在半胱氨酸上的脂肪酸物种的能力。

按照上述协议，我们首先使用acyl-RAC同时检测Jurkat细胞中几种蛋白质的S-化，这是一种不朽的T细胞系，最初来自T细胞白血病患者27的外周血。先前被鉴定为S-acy化99、28、2928,29的调节性T细胞蛋白被选择来证明这种方法的效用。如图2A所示，酪氨酸激酶Lck可以在用中性2 M羟基胺治疗的碱液中...

Watch this Scientific Journal Video about Detection of Protein S-Acylation using Acyl-Resin Assisted Capture at JoVE.com

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Acyl-RAC（阿西尔-树脂辅助捕获）是一种高度敏感、可靠且易于执行的方法，用于检测各种生物样品中半胱氨酸残留物（S-acycyy）的可逆脂改性。

使用阿基-树脂辅助捕获检测蛋白质S-囊肿

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

丙烯酸辅助捕获或Acyl-RAC是一种敏感可靠的方法，可用于检测各种生物样品中的蛋白质S-环化。该协议绕过代谢标签和放射性标签的一些限制，并允许同时检测多种蛋白质的S-细胞化。不仅活细胞，而且原组织和冷冻样品。S-二聚变可以调节各种细胞过程，如蛋白质贩运、血浆膜靶向、信号转导、铁传输和蛋白质-蛋白质相互作用。对于每个实验，使用新鲜准备好的羟基胺溶液，并经过仔细调整的pH值，以确保硫酯键的高效和特定的裂解非常重要。通过演示这种方法，它对于氯仿、甲醇沉淀等步骤至关重要。在此步骤中获得的煎饼形状颗粒应非常小心处理。它可以是脆弱的，在步骤期间样品的部分损失可能导致样品恢复不均匀。演示这个程序的将是萨凡纳·韦斯特，一个来自我们实验室的研究生。要获得细胞解酸，请将感兴趣的细胞收集到锥形离心管中。通过离心去除任何细胞碎片。在5毫升PBS中清洗颗粒。并立即将细胞重新用于600微升新鲜准备的解液缓冲液中。在热摇床中，以每分钟 1，500 转的速度搅拌样品，在 4 摄氏度下搅拌 30 分钟。在通过离心清除溶糖、颗粒洗涤剂和可溶性材料之前。离心结束时，在冰上预冷却的1.5毫升微离心管中收集清除的利沙酸盐。并执行布拉德福德或双辛辛酸测定，根据标准方案估计蛋白质浓度。加入甲醇和氯仿，以2：1的比例进行口水。严格摇动，形成均匀悬浮液，并离心样品，在水相和有机相之间的界面收集蛋白质颗粒。倾斜管，使用针头或凝胶加载尖端吸气尽可能多的溶剂。空气干燥蛋白质颗粒几分钟。在轻轻地将管内装物与600微升甲醇混合之前，注意不要分解颗粒。小心去除甲醇洗涤后，将蛋白质颗粒干燥在长凳上约五分钟。接下来，将蛋白质颗粒重新在200微升2SHB缓冲液中。在热摇床中，温度为42摄氏度，每分钟旋转1500圈。当颗粒溶解时，在2SHB中加入200微升0.2%MMTS。在42摄氏度下孵育蛋白质15分钟，在热摇床中每分钟孵育1500转。在孵育结束时，进行3：4氯仿甲醇沉淀，如证明。要去除MMTS，请将颗粒溶解在100微升新鲜2SHB缓冲液中，并进行涡旋。每次沉淀后，用300微升缓冲液 A 稀释样品。最终沉淀后，将样品溶解在2SHB缓冲液的200微升中，用240微升缓冲液进行稀释。再次测量蛋白质浓度，并留出每个样品中的40微升作为输入控制。将样品分成两个相等的体积 200 微升。并标记管为加羟基胺和减去羟基胺。将 50 微升新鲜准备好的中性二摩尔羟基胺加入 400 毫摩尔的最终浓度添加到加羟基胺管中。和50微升中性二摩尔氯化钠对负对，一个负羟基胺管。然后在每个管中加入30微升的TS珠浆。在室温下旋转管子一到两个小时。在孵化结束时，用缓冲液 A 中的 1%SDS 清洗珠子四次，以去除残留的羟基胺。最后一次洗涤后，用三个温和的一分钟微离心洗洗所有珠子样品。小心吸气的超自然剂和重新暂停珠子在500微升1%SDS在缓冲器 A每次洗涤。上次洗涤后，轻轻旋转珠子，如证明。吸气尽可能多的上一道，而不打扰珠子。为了从珠子中回收蛋白质，将50微升4%SDS样品缓冲液加入每个管中，并在80摄氏度和每分钟1500转的热摇床中孵育样品15分钟。在孵育结束时，让样品冷却后离心，以完全颗粒珠子。然后使用凝胶加载尖端将洗样蛋白转移到新的 1.5 毫升管中。并在SDS-PAGE凝胶上运行样品，通过西方印迹分析感兴趣的蛋白质的S-acylation。酪氨酸激酶Lck可以在用中性二摩尔羟基胺处理的乳酸盐中检测。切开半胱氨酸残留物和脂肪酸莫伊蒂之间的硫酯键。脱衣和与抗体对蛋白质Fyn和LAT的重新剥离表明，丙烯酸树脂辅助捕获测定可用于同时分析多种蛋白质的S-同化。此外，在原小鼠飞溅细胞中，可以很容易地检测到Lck、Fyn和LAT的S-acylation。指示这种修饰在两个物种之间保存。除了识别新型S-甲基化蛋白外，该方法还可用于评估不同生物条件下蛋白质S-甲基化的变化。新识别的S-甲基化蛋白的数量大大增加后，双重增加意味着快速和可靠的技术，如Acyl-RAC。除了识别新型S-甲基化蛋白外，该方法还可用于评估不同生物条件下蛋白质S-甲基化的变化。

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

mRNA Interactome Capture from Plant Protoplasts

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Studying Protein Import into Chloroplasts Using Protoplasts

原生质体在叶绿体中导入蛋白质的研究

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...