Name: ubiquitin chain analysis by parallel reaction monitoring
Uploaded: 2020-06-17T13:00:00
Description: Watch this Scientific Journal Video about Ubiquitin Chain Analysis by Parallel Reaction Monitoring at JoVE.com

作者要感谢Céline Jeanty协助创建细胞颗粒，如代表结果所述，治疗MG-132，以及伊莉丝·莫默茨为她在协议中使用的大肠杆菌颗粒提供。

1. 准备重肽标准
<ol><li>根据所购买的重肽的供应商和质量，重肽需要稀释。该协议使用表1中报告的肽序列，将C+终端氨基酸修改为Lysine（13C615N2-lysine）或精氨酸（13C615N4- 精氨酸）。<ol>
<li>混合重肽，稀释混合物与50%丙酮三酯（ACN），以创建肽混合与每个肽在10 μM。</li>
		<li>创建肽混合物的别名，并存储在 -80 °C。</li>...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Ubiquitin+Binders+to+Decipher+the+Ubiquitin+Code.">Using Ubiquitin Binders to Decipher the Ubiquitin Code.</a> Trends in Biochemical Sciences. 44 (7), 599-615 (2019).</li><li>Newton, 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=Ubiquitin+chain+editing+revealed+by+polyubiquitin+linkage-specific+antibodies.">Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.</a> Cell. 134 (4), 668-678 (2008).</li><li>Spence, J., 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=Cell+cycle-regulated+modification+of+the+ribosome+by+a+variant+multiubiquitin+chain.">Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain.</a> Cell. 102 (1), 67-76 (2000).</li><li>Borr&#224;s, E., Sabid&#243;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+targeted+proteomics?+A+concise+revision+of+targeted+acquisition+and+targeted+data+analysis+in+mass+spectrometry.">What is targeted proteomics? 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L., 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=Iodoacetamide-induced+artifact+mimics+ubiquitination+in+mass+spectrometry.">Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry.</a> Nature Methods. 5 (6), 459-460 (2008).</li><li>Tawa, N. E., Odessey, R., Goldberg, A. 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分析蛋白体内无处不在的状态对各种生物学问题越来越重要。样本无处不在状态的描述不仅必须关注蛋白质无处不在的轮廓，还必须关注这种无处不在的拓扑学。如本文所述，有针对性的MS对这一拓扑学的评估在广泛的生物调查中发挥作用。
应当理解，此处概述的协议提供了全球拓扑概况。使用自下而上的蛋白质组学方法可以确定无处不在的肽，包括无处不在的肽。对无处不...

密切调节蛋白质的功能和稳定性至关重要，因为它们是生物学表型控制的主要驱动因素。蛋白质的功能由两个组成部分构成：其内在多肽序列和任何后翻译修饰（PTM）。已发现各种化学PTM，包括糖化、磷化、乙酰化和甲基化1。1975年，Goldstein等人发现了一种小蛋白质，并将其命名为无处不在的蛋白质。发现无处不在素在蛋白质降解3中很重要。然而，自那时以来，人们已经确定，无处不在的素作为一种信号分子的功能远远超出了蛋白质稳定性的规定。无处不在的信号涉及广泛的其他生物功能，如蛋白质稳定，自噬，细胞循环控制，和蛋白质贩运4。
虽然其他PTM通常是二元的（即，蛋白质被修改或未修改）为给定部位，无处不在的素可以修改蛋白质作为单体或聚合物链，与附加的无处不在本身无处不在。此外，这个多基化链可以在几个道歉中发展，与以前无处不在的无处不在连接到八个链接站点之一5，6。无处不在素通过多步酶过程（图1）转移，其中无处不在素的C-终点站与其七个赖氨酸残留物之一（K06， K11、K27、K29、K33、K48或K63）或N端的先前无处不在（称为M1或线性无处不在）5、6。这种链条拓扑是被修改蛋白质命运的关键。例如，使用 K48 或 K11 链进行修饰会导致蛋白酶体中修改后的蛋白质降解，而线性链对于 NF-kB 信号的激活是必要的。因此，这些链条的相对分布与各种各样的生物学问题有关。
质谱仪 （MS） 的使用对无处不在的链式拓扑分析特别有益，因为它不依赖于基于抗体或亲和力的相互作用7，8， 其中许多具有有限的特异性，不区分不同的链类型。另一种检测可能性是使用转基因无处不在的突变体。在这里，一个特定的莱辛被交换为精氨酸，这不能支持通过无处不在的修改。基板蛋白缺乏无处不在的链形成，然后被解释为特定拓扑9的证据。
基于MS的无所不在蛋白质的鉴定基于这样一个事实，即无处不在的素的C-终点站含有74位的精氨酸残留物，在进行MS分析的样品的蛋白质分析准备过程中，由肌氨酸识别，分离C端双糖氨酸。这种甘油（-GG）仍然附着在基板蛋白赖氨酸残留物的ε-氨基蛋白组。对于无处不在的拓扑分析，该修饰发生在无处不在的七个赖氨酸残留物之一。这创建了一组七个关键肽，携带由 GG 修改的赖氨酸残留物，具体用于每个道歉 （图 2）。例如，使用 K06 拓扑，氨基酸序列中位置 6 的 lysine 将通过 -GG 修改 114 Da 添加到此 lysine 中来防止尝试消化。
MS 对特定预定肽的识别称为靶向蛋白质组学，或更具体地说，定向肽获取10。根据所使用的质谱仪的性能，已经开发出两种方法。这些是选定的反应监测 （SRM），也称为多反应监测 （MRM） 和并行反应监测 （PRM）。SRM 涉及由前体 m/z 和产品离子 m/z 组成的过渡选择。相反，PRM 只需要前体 m/z。选后，对产品离子进行全面的调查扫描。这样做的好处是，在测量11之前，不需要选择合适的产品离子来量化特定的质谱仪。SRM 和 PRM 都可以根据仪器进行安排。调度是分配一个时间窗口的实践，在此期间将包括一个特定的离子进行分析，因为肽在色谱系统的定义保留时间排出。减少在任何给定时间接受审讯的离子数量，增加当时预定的离子的审讯频率，从而提高数据准确性。
一般来说，靶向蛋白质组学在泛素拓扑学中的应用与任何其他靶向蛋白质组学实验相同。然而，两个关键区别是重要的：第一，必须考虑无处不在的链条的稳定性。有许多有效的脱脂酶（DUB），在细胞裂解后迅速降解链条。无处不在的特异性蛋白酶分为两类，硫化物和金属蛋白酶。大多数无处不在的水激光是硫酯酶，并在其活动中心携带一个半胱氨酸残留物。通过碱化这种半胱氨酸残留物，它们可以灭活。因此，使用含有烷基化剂（如 N-乙酰卤化物 （NEM） 和高度变性化学品）的无处不在的稳定缓冲器，并保持样品冷却，对于成功的分析至关重要。其次，与其他有针对性的实验不同，肽的选择是固定的。在典型的靶向实验中，可以选择一种蛋白肽，以获得良好的色谱和电离性能。对于一些拓扑特性的肽，如K48，这些属性是好的，而对于其他人，他们是不太可取的。例如，由于形成拉伸的弹性轮廓，典型逆相设置中的 K33 色谱不佳，K27 肽的电传性能差，其可见性降低了 MS12。
在此协议中，我们描述了如何通过 PRM 对生物样本进行无处不在的拓扑学评估。该过程的示例数据使用MG-132抑制剂治疗在几种不同细胞类型的蛋白酶体的扰动显示。

<table>
	<tbody>
		<tr>
			<td>Acetonitrile (ACN)</td>
			<td>Merck</td>
			<td>100029</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate (ABC)</td>
			<td>Fluka</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Microfuge 16</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroacetamide (CAA)</td>
			<td>Sigma</td>
			<td>22790</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf LoBind</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid (FA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>85178</td>
			<td></td>
		</tr>
		<tr>
			<td>Heavy Peptides</td>
			<td>JPT Peptide Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Dionex</td>
			<td>Ulitimate 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>LC Column</td>
			<td>Thermo Fisher Scientific</td>
			<td>160321</td>
			<td></td>
		</tr>
		<tr>
			<td>Lys C</td>
			<td>Wako</td>
			<td>125-05061</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q-Exactive Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide (NEM)</td>
			<td>ACROS Organics</td>
			<td>156100050</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive Control Chain K48</td>
			<td>Boston Biochem</td>
			<td>UC-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive Control Chain K63</td>
			<td>Boston Biochem</td>
			<td>UC-340-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive Control Chain M1</td>
			<td>Boston Biochem</td>
			<td>UC-710B-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonifier</td>
			<td>Branson sonifier</td>
			<td>SFX 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>Thermomixer Comfort</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigam</td>
			<td>T6508</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td>Thermo Fisher Scientific</td>
			<td>77720</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V1511A</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>51456</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters &#956;Elution C18 plates</td>
			<td>Waters</td>
			<td>186002318</td>
			<td></td>
		</tr>
	</tbody>
</table>

ubiquitin chain analysis by parallel reaction monitoring

评估蛋白组内无处不在的链条道歉的全球概况，有兴趣回答广泛的生物学问题。此处概述的协议利用了在链中加入的无处不在的素尝试消化后留下的二甘氨酸 （-GG） 改性。通过量化这些拓扑特性肽，可以确定每个无处不在的链拓扑的相对丰度。据报道，通过平行反应监测实验量化这些肽所需的步骤考虑到了无处不在的链条的稳定性。与适当的质谱仪设置和数据分析工作流程一起描述了重控制、细胞裂解和消化的准备情况。介绍了一个在无处不在的拓扑中带有扰动的示例数据集，并附有协议优化如何影响结果的示例。通过遵循所概述的步骤，用户将能够在其生物环境中对无处不在的拓扑景观进行全球评估。

为了证明PRM使用无处不在的链分析，选择了三条细胞系：小鼠黑色素瘤细胞系B16，以及两个常见的人类细胞系A549（腺癌肺基上皮细胞）和Hola（宫颈癌细胞）。这些培养在适当的介质中发展到中期，在收获前4小时用0、10或100 mM MG-132进行治疗。MG-132是一种蛋白酶抑制剂，可防止蛋白酶蛋白由蛋白体14降解。鉴于这一点，证明无处不在的链分析是一个适当的测试条件。由此产生的K48?...

Watch this Scientific Journal Video about Ubiquitin Chain Analysis by Parallel Reaction Monitoring at JoVE.com

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

此方法描述了对无处不在的链式拓扑学全球变化的评估。评估通过应用基于质谱的靶向蛋白质组学方法进行。

通过并行反应监测进行无处不在的链分析

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Research

JoVE Journal

Biochemistry

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

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

科研

生物化学

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

rad51 介导的 dna 链交换反应的实时观测

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

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

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 ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

RING-E3型无二体气体在Vitro和Planta中的功能特性

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

Parallel Interrogation of ÃƒÅ½Ã‚Â²-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...