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>
</li>
</ol>2. 样品准备
<ol><li>样本裂解 注:在样品准备过程中，必须考虑无处不在的链条的稳定性。将生物样品和缓冲器保持在 4 °C 的冷却度，并尽快将样品添加到无处不在的稳定缓冲区。<ol>
<li>在水中准备 1 M NH4HCO3 （碳酸氢铵） 的解决方案，并使用 1 M NaOH 将 pH 调整为 8。</li>
		<li>在水中准备 200 mM N-乙酰胺 （NEM） 的解决方案。</li>
		<li>在 50 mM NH4HCO3/10mM NEM 中使用 8 M 尿素准备无处不在的稳定缓冲。 注:8 M 尿素的 5 mL 溶液在尿素饱和溶液中时，由于水的膨胀，需要的水量将大大低于 5mL。 注意:不要准备缓冲超过50°C，因为尿素在高温下形成聚氨酯的倾向。</li>
		<li>将生物样本重新注入无处不在的稳定缓冲区进行调查，以达到0.5微克/微升的蛋白质，并使用适当的方法对细胞进行解体。一种声波方法，由三个10s脉冲组成，SFX 150布兰森声化器设置在70%的强度，每个脉冲之间的冰上休息10，用于本协议中的细胞系。</li>
		<li>离心机样品在桌面离心机的 4 °C 下，在 18，000 x g 下 10 分钟。</li>
		<li>将超自然体转移到新鲜的低蛋白结合微中量管。如果需要，此时样品可存储在-20°C。</li>
	</ol></li>
	<li>样品和控制的准备
	<ol>
<li>如果样品被冷冻，混合（例如，与热混合器），而他们解冻，以迅速溶解尿素。 注意:不要使用温度高于50°C，因为尿素在高温下形成聚氨酯的倾向。</li>
		<li>离心机样品在桌面离心机的 4 °C 下，在 18，000 x g 下 10 分钟。</li>
		<li>将 20 μg 的样品转移到新鲜的低绑定微中福格管中，并利用无处不在的稳定缓冲器将体积调整到 50 μL。</li>
		<li>使用常用素稳定缓冲器 （50 μL） 的正常体积创建负控。在这个样本中，不应存在每个肽的轻版本，从而允许观察由尖刺重肽引起的任何轻污染。此负控也用于保留第 3.1 节中描述的 PRM 的保留时间调度。</li>
		<li>还通过无处不在的稳定缓冲区以及在无处不在的稳定缓冲器 （50 μL） 的正常体积中增加了市售链类型，建立了积极的控制。通常，K63、K48 和 M1 可用。在代表性结果中，使用了20 ng的K48、K63和M1。</li>
	</ol></li>
	<li>
	<li>肽清理
	<ol>
<li>几家制造商提供 C18 清理提示或板。按照制造商对所用产品的说明，在真空离心机中干燥脱硝肽，准备进行MS分析。</li>
	</ol></li>
</ol>3. LC-MS/MS 分析
<ol><li>以不计划的方式对重肽标准进行初始 PRM 分析。<ol>
<li>将干燥样品重新装入 10 μL 的 MS 装载缓冲区 2% ACN/0.05% 三氟乙酸 （TFA）中。</li>
		<li>为 HPLC 配备反相 C18 分析柱（75μm x 15 厘米、2μm 100 °C18 珠），专为纳米流 MS. 形式线性梯度交换 MS 级水，辅以 0.1% FA 和包含 0.1% FA 的 ACN。此处使用了牺牲陷阱柱（75 μm x 2 厘米、3μm、100°C18 珠）来保存分析柱的寿命，但不需要。</li>
		<li>将分析列的输出与高分辨率质谱仪上的纳米 ESI 源进行耦合。</li>
		<li>使用适当的靶向蛋白质组学方法进行质谱仪。例如，在 Q-精确加两个实验方法中，在全 MS 和 PRM 实验之间循环。对于全MS实验，使用分辨率为120，000，AGC目标为1e6和240毫秒作为最大IT。在 PRM 实验中，使用相同的 AGC 目标和最大 IT，分辨率较低，分辨率为 60，000，隔离窗口为 1.2 m/z。</li>
		<li>使用 HPLC 自动采样器将 200 ng 的负控器注入分析列。在分析列上将线性梯度应用于分析列上的样品，在 45 分钟内将 ACN 浓度从 2=25% 增加。 注:由于典型无处不在的拓扑特征肽的亲水性质较低，蛋白质组学的ACN浓度比平常低25%。如果要量化其他肽，可能需要更高的 ACN 浓度才能实现良好的目标分离。</li>
		<li>分析第 4.2 节中描述的调度运行结果，以创建计划包含列表。</li>
	</ol>
</li>
	<li>样本分析<ol>
<li>除使用第 4.2 节中创建的预定包含列表外，运行 3.1 中为调度所描述的其余样本。 注:在正对照后必须运行空白，以确保在后续样本中未检测到上一次运行的结转。</li>
	</ol>
</li>
</ol>4. 软件分析
<ol><li>为已使用的 MS 创建适当的格式包含列表。该协议使用开源软件天际线（版本19.1.0.193）（https://skyline.ms），它提供了有据可查的支持和帮助。使用出口隔离列表设施，可为多个质谱仪创建包含列表。<ol>
<li>打开新的天际线文档。</li>
		<li>选择重同位素修饰，以适当重拓扑特性肽。在设置中|修改选项卡下的肽设置，单击名称字段以查看潜在的修改。选择基于购买的重肽的同位素状态。在此示例中，在 C 终点站使用携带重 Lysine（13C615N2- lysine） 或重精氨酸（13C615N4- 精氨酸） 的合成肽。适当的标签为:"标签:13C （6）15N （2） （C-术语 K）"和"标签:13C （6）15N （4） （C 学期 R）"。</li>
		<li>选择"设置|过渡。过滤器选项卡下包括前体费用2，3，4。 </li>
		<li>通过选择编辑插入肽 |插入|肽。</li>
		<li>填写相关领域，包括拓扑特性的肽序列和蛋白质名称（如"链–K63"）。</li>
		<li>展开 目标面板中 现在显示的每个肽。删除未所需的充电状态。首选的电荷状态和碰撞能量，可能因使用质谱仪而异，因为每个拓扑特性肽都显示在 表 1 中。</li>
		<li>对于每个拓扑特性的肽（M1除外，其中包含GG的序列）右键单击序列并选择 修改。通过单击名称字段并选择<显示所有。。。>在选择"甘格利（K）"之前。</li>
		<li>导出隔离列表 文件|出口|隔离列表，选择仪器类型，并将方法类型设置为 标准。选择 "确定 "将打开一个提示，以保存可用于创建 PRM 方法的 *.csv 文件。</li>
	</ol></li>
	<li>使用开源 Skyline 软件在此处解释的重肽调度运行创建计划包含列表。<ol>
<li>创建第 4.1 节中描述的目标列表。</li>
		<li>通过选择文件导入 3.1 中的调度运行 |导入|结果.</li>
		<li>查看标识。通过单击每个重肽条目的质量，可以观察到每个肽的重信号。峰值的正确识别通常自动确定，但软件可能需要手动固化，通过点击并拖动到 x 轴下来选择峰值。</li>
		<li>为重变种选择的保留时间也应用于轻型版本，从而为 PRM 创建了时间表。可在"设置"下修改调度窗口 | 使用 预测 选项卡的肽设置，通过更改 时间窗口。</li>
		<li>现在可以使用文件导出计划包含列表 |出口|隔离 列表和选择适当的仪器类型和设置方法类型到 预定。</li>
	</ol></li>
	<li>分析数据。<ol>
<li>以与第 4.2 节中的调度运行分析相同的方式导入样品。</li>
		<li>在完全导入所有样品后，建议进行过渡策展。这是消除干扰或信号与噪声比率差的过渡的过程。图 4中显示了策展前后的色谱图示例。</li>
		<li>现在可以通过右键单击相关图形和选择 副本数据 或选择文件来导出数据 |出口|结果.</li>
	</ol></li>
</ol>

<ol><li>Khoury, G. A., Baliban, R. C., Floudas, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteome-wide+post-translational+modification+statistics%3A+frequency+analysis+and+curation+of+the+swiss-prot+database%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database.</a> Scientific Reports. 1, 90(2011).</li>
<li>Goldstein, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+a+polypeptide+that+has+lymphocyte-differentiating+properties+and+is+probably+represented+universally+in+living+cells%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 72 (1), 11-15 (1975).</li>
<li>Glickman, M. H., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ubiquitin-proteasome+proteolytic+pathway%3A+Destruction+for+the+sake+of+construction%5BTitle%5D)+AND+%22Physiol.+Rev%22%5BJournal%5D)">The ubiquitin-proteasome proteolytic pathway: Destruction for the sake of construction.</a> Physiol. Rev. 82, 373-428 (2002).</li>
<li>Pickart, C. M., Eddins, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ubiquitin%3A+structures%2C+functions%2C+mechanisms%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta+%28BBA%29+-+Molecular+Cell+Research%22%5BJournal%5D)">Ubiquitin: structures, functions, mechanisms.</a> Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1695 (1-3), 55-72 (2004).</li>
<li>Akutsu, M., Dikic, I., Bremm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ubiquitin+chain+diversity+at+a+glance%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Ubiquitin chain diversity at a glance.</a> Journal of Cell Science. 129 (5), 875-880 (2016).</li>
<li>Komander, D., Rape, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Ubiquitin+Code%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">The Ubiquitin Code.</a> Annual Review of Biochemistry. 81 (1), 203-229 (2012).</li>
<li>Mattern, M., Sutherland, J., Kadimisetty, K., Barrio, R., Rodriguez, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+Ubiquitin+Binders+to+Decipher+the+Ubiquitin+Code%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">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/pubmed?term=((Ubiquitin+chain+editing+revealed+by+polyubiquitin+linkage-specific+antibodies%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Cell+cycle-regulated+modification+of+the+ribosome+by+a+variant+multiubiquitin+chain%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain.</a> Cell. 102 (1), 67-76 (2000).</li>
<li>Borràs, E., Sabidó, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+is+targeted+proteomics%3F+A+concise+revision+of+targeted+acquisition+and+targeted+data+analysis+in+mass+spectrometry%5BTitle%5D)+AND+%22PROTEOMICS%22%5BJournal%5D)">What is targeted proteomics? A concise revision of targeted acquisition and targeted data analysis in mass spectrometry.</a> PROTEOMICS. 17 (17-18), (2017).</li>
<li>Lesur, A., Domon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+high-resolution+accurate+mass+spectrometry+application+to+targeted+proteomics%5BTitle%5D)+AND+%22PROTEOMICS%22%5BJournal%5D)">Advances in high-resolution accurate mass spectrometry application to targeted proteomics.</a> PROTEOMICS. 15 (5-6), 880-890 (2015).</li>
<li>Longworth, J., Dittmar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+Ubiquitin+Chain+Topology+by+Targeted+Mass+Spectrometry%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Assessment of Ubiquitin Chain Topology by Targeted Mass Spectrometry.</a> Methods in Molecular Biology. 1977, Clifton, N.J. 25-34 (2019).</li>
<li>Nielsen, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iodoacetamide-induced+artifact+mimics+ubiquitination+in+mass+spectrometry%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitors+of+the+proteasome+reduce+the+accelerated+proteolysis+in+atrophying+rat+skeletal+muscles%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.</a> The Journal of Clinical Investigation. 100 (1), 197-203 (1997).</li>
<li>Kim, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+and+quantitative+assessment+of+the+ubiquitin-modified+proteome%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Systematic and quantitative assessment of the ubiquitin-modified proteome.</a> Molecular Cell. 44 (2), 325-340 (2011).</li>
<li>MacLean, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+Collision+Energy+Optimization+on+the+Measurement+of+Peptides+by+Selected+Reaction+Monitoring+%28SRM%29+Mass+Spectrometry%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Effect of Collision Energy Optimization on the Measurement of Peptides by Selected Reaction Monitoring (SRM) Mass Spectrometry.</a> Analytical Chemistry. 82 (24), 10116-10124 (2010).</li>
</ol>

作者声明没有相互竞争的利益。

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。对无处不在的肽无处不在的观察可以确定链拓扑学，但与无处不在素目标的联系丢失。因此，无法确定任何特定蛋白质的特定链拓扑学，但全球拓扑特征是衍生的。该方法还可以与浓缩战略相结合，以提供更有针对性的结果。这种丰富必须考虑到链条的稳定性和浓缩后链条的丰富性。链式拓扑学的检测受富集策略提供的纯度的限制，因为不可能区分与污染物蛋白相关的无处不在的拓扑学与与兴趣蛋白相关的拓扑。该协议的关键步骤，促进成功的分析，是平衡保存无处不在的链，同时消化无处不在的蛋白质。这是具有挑战性的，因为链条断裂的倾向加上无处不在本身对酶消化的抵抗力。此外，鉴于某些拓扑特征肽对液相色谱耦合质谱分析的有利性质较低，需要添加支持基质并仔细考虑 MS 条件。
虽然我们在本协议中概述了同时评估所有无处不在的链式的一种手段，但也可以根据感兴趣的特定链拓扑学调整协议的几个方面。操纵色谱条件或改变重肽浓度可以提高调查的准确性，改善特定链式拓扑的结果，并根据建议的生物学问题调整调查。

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 &amp;mu;Elution C&lt;sub&gt;18&lt;/sub&gt; 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链增加可以从 图3中看到。蛋白酶抑制剂治疗诱导增加K48链15。
如介绍中所述，与 SRM 或 MRM 不同，PRM 在选择前体离子后执行完整的产品离子扫描。虽然这意味着产品离子不需要在运行前指定，但它们仍应在运行后进行策划。固化是选择真正代表预期肽的过渡的过程。 图4 显示了 K48 在策划前后的产品离子色谱图。具有可能因干扰而具有不一致回旋轮廓的信号的产品离子被移除。低强度产品离子随后被移除，因为信号与噪声比对此类转换不太有利。在策展过程中选择的最佳过渡通常在实验之间是一致的，并且可能因所使用的质谱仪、色谱条件、分析设置和样本的生物背景而有所不同。因此，对于每项调查，都需要适当地制定过渡计划。还需要在包含更多过渡以及技术复制和更清洁的数据之间取得平衡，以进行量化。
在这个实验中，每个已识别的无处不在的链条的典型产品离子色谱图都显示在 图5中。K27 和 K29 在这里被省略，因为在这些生物条件下，信号低于检测。此处所示的 K33 的阐释配置文件比 PRM 分析通常接受的要广泛得多。然而，在无处不在的链分析中，不可能选择具有更好弹性特征的肽的替代选择，并且必须根据此配置文件进行解释。如果 K33 具有特殊意义，则改变色谱条件可能会改善这种链式的量化，但可能会损害其他链条类型。
在设计 PRM 实验时，最好通过增加信号到噪声来最大化用于定量的产品离子的信号。碰撞能量的优化（即质谱仪用来将前体肽离子碎片到产品离子上的能量）是改善信号的一种手段。碰撞能量范围从14–28被应用于重复注入一个样本，结果观察到的峰值区域K63和M1显示在 图6中。可见，对于K63来说，26的碰撞能量是最佳的，而对于M1来说，18的低能量是理想的。每个拓扑特性肽的碰撞能量和一系列未经修改的无处不在的片段在 表1中显示。这些碰撞能量可能需要根据所使用的质谱仪和碎片化方法进行优化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60702/60702fig01.jpg"> 图1:无处不在的结合级联的示意图表示。无处不在素首先以 ATP 依赖的方式受 E1 酶的约束。然后，激活的无处不在素被转移到E2酶中，然后由E3-ligase加入，将无处不在的素转移到基板蛋白中。这个过程重复多次，直到基板蛋白形成无处不在的链条。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60702/60702fig01large.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/60702/60702fig02.jpg"> 图2:图示显示无处不在的拓扑特性肽的衍生。后尝试消化上游结合部位与下游无处不在的-GG C终端相连。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60702/60702fig02large.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/60702/60702fig03.jpg"> 图3:MG-132治疗下的K48链对比小鼠细胞系B16和两个人类细胞系A459和 HeLa。在A459中，MG-132浓度的增加导致K48链条的稳定。B16 和 HeLa 细胞在治疗中出现增加，为 10 mM 或 100 mM MG-132。观察到的从10mM到100mM的减少可以解释为B16和 HeLa细胞对MG-132的敏感性增加，导致细胞死亡。错误栏显示过渡的标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60702/60702fig03large.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/60702/60702fig04.jpg"> 图4:M1拓扑特性肽在进行干扰或低强度过渡前后的产品离子色谱图。x 轴上的保留时间以分钟数显示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60702/60702fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60702/60702fig05.jpg"> 图5:各种拓扑特征肽的典型产品离子色谱以及两种非改性无处不在的肽。在相关峰值的顶点上方报告每肽在几分钟内的释放时间并观察到质量错误。点缀线也显示确定峰值区域的窗口。x 轴上的保留时间以分钟数显示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60702/60702fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60702/60702fig06.jpg"> 图6:在一系列规范化碰撞能量中观察到K63和M1的峰值区域。不同的颜色表示每个过渡贡献的离子强度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60702/60702fig06large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:font-size="8px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>链式类型</td>
			<td>拓扑特征肽</td>
			<td>充电状态</td>
			<td>标准化碰撞能量</td>
		</tr>
<tr>
<td>K06</td>
			<td>姆基夫维克</td>
			<td>3</td>
			<td>18</td>
		</tr>
<tr>
<td>K11</td>
			<td>特克格克 [Gg] 标题</td>
			<td>3</td>
			<td>18</td>
		</tr>
<tr>
<td>K27</td>
			<td>标题</td>
			<td>3</td>
			<td>18</td>
		</tr>
<tr>
<td>K29</td>
			<td>阿克格伊克</td>
			<td>2</td>
			<td>22</td>
		</tr>
<tr>
<td>K33</td>
			<td>伊克德克 [GG] 埃吉普德克</td>
			<td>3</td>
			<td>18</td>
		</tr>
<tr>
<td>K48</td>
			<td>利法格克[格]克莱德格尔</td>
			<td>3</td>
			<td>24</td>
		</tr>
<tr>
<td>K63</td>
			<td>特尔斯迪尼克 [GG] 埃斯特勒夫尔</td>
			<td>4</td>
			<td>26</td>
		</tr>
<tr>
<td>M1（线性）</td>
			<td>格姆基夫克</td>
			<td>2</td>
			<td>18</td>
		</tr>
<tr>
<td>非改性乌布秋伊汀</td>
			<td>埃斯特勒夫尔</td>
			<td>2</td>
			<td>26</td>
		</tr>
<tr>
<td>非改性乌布秋伊汀</td>
			<td>标题</td>
			<td>2</td>
			<td>18</td>
		</tr>
<tr>
<td>非改性乌布秋伊汀</td>
			<td>特尔斯迪尼克</td>
			<td>2</td>
			<td>18</td>
		</tr>
</tbody></table>表1:人类无处不在的拓扑特征肽的序列，最明显的电荷状态，以及有利的规范化碰撞能量。

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 ...

ubiquitin-chain-analysis-by-parallel-reaction-monitoring

Nanyang Technological University

Research

JoVE Journal

Biochemistry

3.5K 次观看.  Luxembourg Institute of Health. 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。

视频: Ubiquitin Chain Analysis by Parallel Reaction Monitoring

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 ...

科研

生物化学

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

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

E3泛素连接酶在哺乳动物细胞裂解物中底物泛素化的评价

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

测量泛素化、SUMO 化和 ADP 核糖基化 DNA-蛋白质交联

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

作者聚焦: DNA 蛋白交联及其翻译后修饰的定量检测  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

<meta charset="utf8"></head><body>使用切割测定评估神经发育障碍中的 DUB 变异

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

<meta charset="utf8"></head><body>通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. ...

通过并行反应监测进行无处不在的链分析

摘要

探索更多视频

通过肽浓缩和质谱检测蛋白质泛化位点检测

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

蛋白泛素化的检测

E3泛素连接酶在哺乳动物细胞裂解物中底物泛素化的评价

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性