Name: evaluation of protein&#8211;protein interactions using an on-membrane digestion technique
Uploaded: 2019-07-19T13:30:00
Description: Watch this Scientific Journal Video about Evaluation of ProteinÃ¢â‚¬â€œProtein Interactions using an On-Membrane Digestion Technique at JoVE.com

这项研究部分得到了日本科学促进协会KaKENHI赠款号17K09869(至AM)、日本科学促进会KaKENHI赠款号15K09418(至TM)的支持,这是来自Kanehara Ichiro医学科学基金会的一笔研究补助金。和苏津纪念基金会的研究补助金(全部给TM)。

1. 免疫沉淀
注:我们使用非硫酸钠(SDS)溶解液缓冲液和柠子酸盐洗脱,如下各节所述。然而,使用替代的内部免疫沉淀技术可能也适用于制备LC-MS/MS样本。
<ol><li>转染培养细胞与载体编码表位标记单独或融合蛋白。为了获得代表性数据,使用载体单独编码绿色荧光蛋白 (GFP)或使用阳离子脂质体处理 GFP 融合 Capn6(calpain-6 基因),传输 J774 细胞(1 x 10 6)。</li>
	<li>抗体与磁珠结合?...

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-translational+protein+folding:+progress+and+methods.">Co-translational protein folding: progress and methods.</a> Current Opinion in Structural Biology. 42, 83-89 (2017).</li><li>Miyazaki, T., Miyazaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+protein+catabolism+in+atherosclerotic+vascular+inflammation.">Defective protein catabolism in atherosclerotic vascular inflammation.</a> Frontiers in Cardiovascular Medicine. 4, 79 (2017).</li><li>Miyazaki, T., Miyazaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+calpain+proteolytic+systems+underlies+degenerative+vascular+disorders.">Dysregulation of calpain proteolytic systems underlies degenerative vascular disorders.</a> Journal of Atherosclerosis and Thrombosis. 25 (1), 1-15 (2018).</li><li>Steckelberg, A. L., Boehm, V., Gromadzka, A. M., Gehring, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CWC22+connects+pre-mRNA+splicing+and+exon+junction+complex+assembly.">CWC22 connects pre-mRNA splicing and exon junction complex assembly.</a> Cell Reports. 2 (3), 454-461 (2012).</li><li>Turriziani, B., von Kriegsheim, A., Pennington, S. 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B., Tabb, D. L., Hervey, W. J., Pan, C., Hurst, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+specific+trypsin+digestion+of+microgram+to+nanogram+quantities+of+proteins+in+organic-aqueous+solvent+systems.">Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems.</a> Analytical Chemistry. 78 (1), 125-134 (2006).</li><li>Miyazaki, T., Miyazaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+calpain+proteolytic+systems+in+macrophage+cholesterol+handling.">Emerging roles of calpain proteolytic systems in macrophage cholesterol handling.</a> Cellular and Molecular Life Sciences. 74 (16), 3011-3021 (2017).</li><li>Miyazaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain-6+confers+atherogenicity+to+macrophages+by+dysregulating+pre-mRNA+splicing.">Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing.</a> Journal of Clinical Investigation. 126 (9), 3417-3432 (2016).</li><li>Tonami, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain-6,+a+microtubule-stabilizing+protein,+regulates+Rac1+activity+and+cell+motility+through+interaction+with+GEF-H1.">Calpain-6, a microtubule-stabilizing protein, regulates Rac1 activity and cell motility through interaction with GEF-H1.</a> Journal of Cell Science. 124 (Pt 8), 1214-1223 (2011).</li><li>Rodnina, M. 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我们之前已经描述了使用LC-MS/MS在膜上消化技术6之前,在氧化的低密度脂蛋白中对脂蛋白B-100的氧化修饰的分析。在本研究中,我们将该技术与免疫沉淀相结合,并确定了几种与钙痛-6相关的蛋白质。这种新技术代表了筛选候选相关蛋白质的便捷方法。Calpain-6是钙蛋白酶家族11的非蛋白解体成员,据报道,它通过其蛋白质-蛋白质相互作用12、13<...

虽然蛋白质在生物体中起着构成作用,但它们在细胞内环境中不断被合成、加工和降解。此外,细胞内蛋白质经常在物理和生物化学上相互作用,这会影响一个或两个1,2,3的功能。例如,将与plissome相关的蛋白同源性CWC22与真核转化启动因子4A3(eIF4A3)直接结合是组装外子结复合物4所必需的。与此一致,一种对CWC22缺乏亲和力的eIF4A3突变体未能促进外子结复合驱动的mRNA拼接4。因此,蛋白质相互作用的研究对于准确理解生理调节和细胞功能至关重要。
质谱学(MS)的最新进展已应用于蛋白质-蛋白质相互作用的综合分析。例如,内源性蛋白质或外源引入标记蛋白及其相关蛋白的共沉淀,然后进行MS分析,能够识别新的蛋白质相互作用5。然而,MS/MS分析的一个主要瓶颈是蛋白质样品的胰蛋白消化不良恢复。用于对细胞解物进行蛋白体分析,通常采用凝胶内和膜内消化技术来制备MS/MS样品。我们之前将凝胶内消化程序与膜内消化技术6进行了比较,并表明后者与更好的序列覆盖率有关。聚苯乙烯二氟化酶 (PVDF) 膜可能适合于此目的,因为它机械坚固,耐高浓度有机溶剂7、8,允许固定的酶消化蛋白质在存在80%醋酸酯9。此外,膜上的固定可以诱导目标蛋白的构象变化,导致胰体消化效率的提高10。因此,在本文中,我们描述了使用膜内消化技术对蛋白质相互作用进行免疫沉淀-LC/MS/MS分析的使用。这种简单的方法便于分析蛋白质-蛋白质相互作用,即使在非专业实验室。

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Wako</td>
			<td>014-00386</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Wako</td>
			<td>030-05525</td>
			<td></td>
		</tr>
		<tr>
			<td>DiNA</td>
			<td>KYA Tech Co.</td>
			<td></td>
			<td>nanoflow high-performance liquid chromatography</td>
		</tr>
		<tr>
			<td>DiNa AI</td>
			<td>KYA Tech Co.</td>
			<td></td>
			<td>nanoflow high-performance liquid chromatography equipped with autosampler</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Nacalai tesque</td>
			<td>14112-94</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads protein G</td>
			<td>Thermo Fisher Scientific</td>
			<td>10003D</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Wako</td>
			<td>066-00461</td>
			<td></td>
		</tr>
		<tr>
			<td>HiQ Sil C18W-3</td>
			<td>KYA Tech Co.</td>
			<td>E03-100-100</td>
			<td>0.10mmID * 100mmL</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Wako</td>
			<td>095-02151</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000008</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Colors A.v. Monoclonal Antibody (JL-8)</td>
			<td>Clontech</td>
			<td>632380</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4HCO3</td>
			<td>Wako</td>
			<td>018-21742</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40</td>
			<td>Sigma</td>
			<td>N6507</td>
			<td>poly(oxyethelene) octylphenyl ether (n=9)</td>
		</tr>
		<tr>
			<td>peptide standard</td>
			<td>KYA Tech Co.</td>
			<td>tBSA-04</td>
			<td>tryptic digests of bovine serum albumin</td>
		</tr>
		<tr>
			<td>PP vial</td>
			<td>KYA Tech Co.</td>
			<td>03100S</td>
			<td>plastic sample tube</td>
		</tr>
		<tr>
			<td>Protease inhibitor cooctail</td>
			<td>Sigma</td>
			<td>P8465</td>
			<td></td>
		</tr>
		<tr>
			<td>ProteinPilot software</td>
			<td>Sciex</td>
			<td>5034057</td>
			<td>software for protein identification</td>
		</tr>
		<tr>
			<td>Sequencing Grade Modified Trypsin</td>
			<td>Promega</td>
			<td>V5111</td>
			<td>trypsin</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic dihydrate</td>
			<td>Sigma</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>TFA</td>
			<td>Wako</td>
			<td>206-10731</td>
			<td></td>
		</tr>
		<tr>
			<td>trap column</td>
			<td>KYA Tech Co.</td>
			<td>A03-05-001</td>
			<td>0.5mmID * 1mmL</td>
		</tr>
		<tr>
			<td>TripleTOF 5600 system</td>
			<td>Sciex</td>
			<td>4466015</td>
			<td>Hybrid quadrupole time-of-flight tandem mass spectrometer</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Wako</td>
			<td>207-06275</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Wako</td>
			<td>160-21211</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of protein&#8211;protein interactions using an on-membrane digestion technique

许多细胞内蛋白质根据细胞内和细胞外情况进行物理交互。事实上,细胞功能很大程度上依赖于细胞内蛋白质与蛋白质的相互作用。因此,关于这些相互作用的研究对于促进对生理过程的理解是必不可少的。相关蛋白质的共沉淀,然后进行质谱分析(MS),能够识别新的蛋白质相互作用。在这项研究中,我们详细介绍了免疫沉淀-液相色谱(LC)-MS/MS分析与膜内消化相结合,用于蛋白质与蛋白质相互作用的分析。该技术适用于粗免疫沉淀物,可提高蛋白组分析的通量。标记重组蛋白是使用特定抗体沉淀的;其次,在聚氯丁二烯膜片片上被斑点的免疫沉淀物进行还原烷基化。在胰蛋白酶化后,使用LC-MS/MS对消化的蛋白质残留物进行分析。因此,该方法对新型蛋白质与蛋白质相互作用的表征是方便和有用的。

通过上述程序,使用LC-MS/MS(图1)对免疫沉淀物进行了分析。排除外源性蛋白质(来自其他物种和IgG的蛋白质)后,在降钙-6相关免疫沉淀物中识别了17种蛋白质(表1),在GFP相关免疫沉淀物中识别了15种蛋白质(表 2)。在降钙-6和GFP相关蛋白中,有11种在两种免疫沉淀物中被识别(图2)。一旦排除这些和降钙酶-6本身,5种?...

Watch this Scientific Journal Video about Evaluation of ProteinÃ¢â‚¬â€œProtein Interactions using an On-Membrane Digestion Technique at JoVE.com

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

在这里,我们提出了一个方案,用于制备质谱样本的膜上消化技术。该技术有助于方便分析蛋白质与蛋白质的相互作用。

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

研究蛋白质-蛋白质相互作用对于理解多个领域的生理过程是必不可少的。在这段视频中，我们介绍了膜上消化技术，使研究人员能够方便地对MS的结合蛋白进行全面分析。该技术的主要优点是，由于多丙烯酰胺凝胶电泳分离蛋白质，因此可以提高粗免疫沉淀物蛋白质分析的通量。通过将抗GSP抗体与500微升磷酸酸盐缓冲液中的珠子混合，开始与磁珠结合抗体。在室温下以 50 RPM 旋转混合物一小时。然后用含有1%聚糖酸酯20的柠檬酸盐缓冲液洗三次联结珠。根据手稿指示对转染细胞进行精做。刮盘，将利沙酸盐转移到1.5毫升试管中。然后以15，000xg的离心清除酸盐5分钟，然后收集上清液。用500微升的柠檬酸盐缓冲液洗三次，准备未标记的磁珠。最后洗涤后，取出缓冲液，将细胞化到珠子中。在室温下以 50 RPM 旋转混合物 30 分钟。在磁性支架上进行磁分离并收集细胞分离。要将目标蛋白与免疫球蛋白结合珠子结合，请将珠子放在磁性支架上五分钟。丢弃柠檬酸盐缓冲液并将细胞酸盐添加到珠子中。以 50 RPM 的速度旋转混合物一小时。为了将目标蛋白与自由非靶蛋白分离，用含有1%聚糖酸20的500微升柠檬酸盐磷酸盐缓冲液洗三次珠子。最后洗涤后，加入30微升柠檬酸盐缓冲液，让珠子在室温下坐5分钟，以洗净目标蛋白质。要准备膜，请使用手术剪刀将 PVDF 膜切割成 3 毫米 3 毫米，这些剪刀在使用前立即进行清洁。将膜片放在铝箔上，在每片中加入两到五微升乙醇。在膜完全干燥之前，将蛋白质洗出的两到五微升添加到每片，然后风干，直到膜表面变得哑光。然后将膜转移到1.5毫升管中，并在4摄氏度下储存。如果立即使用，加入2至30微升乙醇，使它们亲水。用移液器去除乙醇，但在膜干燥之前，将200微升的DTT反应溶液完全添加到每个管中，并在56摄氏度下孵育一小时。孵育后，用300微升的碘多乙酰胺溶液代替反应溶液，在黑暗中孵育45分钟。然后用蒸馏水洗两次膜，一次用2%的乙酰酸十分，用涡流至少洗涤十秒钟。使用与透明相关的乙酰胺可能极其危险。执行膜上消化程序时，应始终佩戴口罩、眼镜和手套。根据手稿方向准备 trypsin，并在每个膜上加入 100 微升的 trypsin 反应溶液。在37摄氏度下孵育，用于消化。第二天，将反应溶液转移到一个干净的1.5毫升管中，并在膜中加入100微升的洗涤液。在60摄氏度下孵育膜两小时，然后收集洗涤液，与反应溶液混合。再在膜上加入100微升的洗涤液，然后对膜进行10分钟的声波化。然后收集洗涤液，并混合与反应溶液。用一块实验室膜盖住试管，用针头在薄膜上打小孔。使用真空浓缩器干燥溶液，将残留物溶解在 10 微升 2%甲酸中。将管以12，000 x g离心3分钟，然后将上看液转移到样品管中。使用与纳米 LC/HPLC 系统相连的质谱仪分析样品。利用该协议，在Calpain-6相关免疫沉淀物中确定了17种蛋白质，在GP相关免疫沉淀物中确定了15种蛋白质，在两种免疫沉淀物中确定了11种蛋白质。需要注意的是，检测至少一个高保真肽片段对于候选蛋白的阳性识别是必需的。在GP表达的脂肪以及血毒蛋白、外源蛋白和核糖体蛋白中沉淀的蛋白质从列表中被省略。用蒸馏水和2%乙酰三叶酸在试消化前清洗膜是膜上消化过程中最重要的一步。采用这种方法，可以通过进行免疫沉淀和西印分析等实验来确认靶蛋白的共沉淀。这个简单的方法使研究人员能够通过方便和高灵敏度的分析来探索新型的蛋白质-蛋白质相互作用。

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Unit 1

Protein Function

SCIENTISTS IN ACTION

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

对于拟肽与他们抗利什曼原虫活性混合赖氨酸型/精氨酸型单体及评价合成的有效方法

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In 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 ...

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

映射的适体对ATP的结合部位使用MicroScale程序热泳

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

膜蛋白对洗涤剂敏感相互作用的检测

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

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 ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...