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>

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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...

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链...

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

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

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

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

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

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

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

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

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

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

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

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

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

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

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 ...

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

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

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 ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

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 ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.