作者感谢清李的录音帮助。这项研究得到了来自德州理工大学健康科学中心的启动基金的支持, 以及由翻译神经科学和治疗中心 (CTNT) 授予 PN-CTNT 2017-05 AKHRJDHW A.L.K.;部分由 NIH 授予 R01AI099380 对 K.Z. 詹姆斯 c. 霍夫曼和克里斯汀 r 巴卡是喜赛 (干细胞教育 & 研究的整合中心) 学者并且由节目支持。

所有动物的治疗和组织的处理获得的研究是根据协议的机构动物护理和使用委员会在德州理工大学健康科学中心按照国家研究所健康动物福利准则, 96005 号议定书。请牺牲脊椎动物, 根据动物保育和使用委员会的指导方针准备组织。如果缺乏这样一个委员会, 请参考国家卫生研究院动物福利指南。成人 (> 60 天) C57BL/6 小鼠使用。所有动物和组织都是根据美国德州理工大学卫生科学中心的机构动物护理和使用委员会批准的协议获得的, 根据国家卫生动物福利研究所的准则。对于安乐死, 一只老鼠被放在一个小房间里, 空气逐渐偏移, 大约有30% 二氧化碳麻醉, 并将动物的痛苦降到最低。在呼吸停止后, 我们用颈椎脱位来确认动物在收割组织前的死亡。
注意: 所有与活的利什曼原虫和培养的人类细胞的工作都是在 BSL-2 认证实验室的生物安全柜中完成的。
1. 从利什曼原虫少校、培养的人体细胞和小鼠组织中制备细胞质裂解物
注: 不同来源材料的裂解制剂有几个不同之处。其他步骤, 包括蔗糖梯度准备和 polysomal 分馏是相同的, 不依赖于样品来源。
<ol><li>
利什曼原虫主要细胞质裂解制剂
	<ol>
<li>接种利什曼原虫少校(FV1 应变) 细胞在30毫升 1x M199 中29含有10% 胎牛血清 (血清) 和青霉素/链霉素混合物 (100 单位和100微克/毫升相应) 在密度 1x105细胞/毫升. 注意: 涉及利什曼原虫主要单元格的所有步骤都必须在生物安全柜中进行。</li>
		<li>将细胞放置在孵化器中, 并将其生长在27摄氏度, 直到对数相 (中间的日志对应于 5x106细胞/mL)。它通常需要两天的时间来成长。</li>
		<li>将放线菌酮添加到利什曼原虫主要区域性, 最终集中100微克/毫升以在翻译的基因上逮捕核糖体。将细胞放回孵化器中10分钟, 在27摄氏度。</li>
		<li>放线菌酮治疗完成后, 将细胞转移到50毫升圆锥管, 并将其旋转 1800 x g 和4°c, 以8分钟为单位丢弃上清。</li>
		<li>用30毫升 Dulbecco 磷酸盐缓冲盐水 (DPBS) 冲洗细胞。离心机在 1800 x g 和4°c 为8分钟。</li>
		<li>放弃上清。并用重悬细胞在1毫升的 DPBS。</li>
		<li>整除细胞, 并将其与3.5% 甲醛溶液混合。</li>
		<li>通过 hemocytometer 计数细胞并确定它们的浓度。将所需的细胞数转移到离心管中。从 0.5x108-2x108细胞/mL 中制备的裂解液足够用于一个蔗糖梯度加载。</li>
		<li>旋转细胞在 1800 x g 和4°c 为8分钟. 丢弃上清。</li>
		<li>并用重悬在1毫升含有蛋白酶抑制剂和 RNase 抑制剂 (20 毫米 HEPES) 的裂解缓冲液中, 将细胞颗粒放在冰上, pH 值 7.4, 100 毫米氯化钾, 10 毫米氯化镁2, 2 毫米, 1% NP-40, 1x 蛋白酶抑制剂鸡尾酒 (无 EDTA), 200 单位/毫升 RNase 抑制剂)。</li>
		<li>通过23口径针三倍的裂解液。在通过针后, 裂解液应变得透明。</li>
		<li>离心机在 11200 x g 和4°c 为10分钟澄清裂解。将澄清的裂解液转移到新鲜的管子上, 并将其保存在冰上直到蔗糖梯度离心。</li>
		<li>收集 400-500 ul 的裂解剂作为输入 (稍后分析), 冻结它在液氮中的未来蛋白质分析或添加 rna 纯化试剂冷冻前的 rna 分析。</li>
	</ol>
</li>
	<li>
人 HeLa 细胞培养的细胞质裂解制剂
	<ol>
<li>将 HeLa 细胞分裂成20毫升的 DMEM 培养基, 其中含有10% 的血清和青霉素/链霉素混合物 (100 个单位和100微克/毫升相应), 细胞计数 2x105细胞/毫升在15厘米板中。</li>
		<li>生长 HeLa 细胞在37°c, 5% CO2为 20-24 h. 根据制造商的协议进行质粒 DNA 转染。</li>
		<li>在37°c、5% CO2转染后, 将细胞传播24小时。</li>
		<li>添加放线菌酮到生长的 HeLa 细胞到最后浓度的100微克/毫升, 以逮捕核糖体在翻译基因和孵化细胞为10分钟37摄氏度, 5% CO 2.吸入培养基。用冷 DPBS 在冰上洗两次细胞。</li>
		<li>添加 500 ul 的裂解缓冲 (20 毫米 HEPES-KOH pH 7.4, 100 毫米氯化钾, 5 毫米氯化镁2, 1 毫米, 0.5% NP-40, 1x 蛋白酶抑制剂鸡尾酒 (无 EDTA), 200 单位/毫升 RNase 抑制剂或1毫克/毫升肝素) 到板上, 并刮在冰上的细胞。</li>
		<li>将裂解细胞转移到离心管。根据样品体积的增加, 将 NP-40 的浓度调整为0.5% 和氯化镁2到5毫米。</li>
		<li>通过23口径针3-6 倍的裂解液。</li>
		<li>旋转 11200 x g 和4°c 8 分钟, 以澄清裂解。离心后, 将上清液转移到新管上。使用分光光度计评估细胞裂解效率, 并确定在梯度上加载的样本量。添加 10 ul 样品到0.5 毫升的0.1% 月桂酸钠 (SDS)。空白对 0.1% SDS。测量吸光度在 260 nm。预期吸光度值约为15-20 单位/毫升。</li>
		<li>在蔗糖梯度离心前稀释所有与裂解缓冲剂相同的吸光度值。将样品保存在冰上直到蔗糖梯度离心。</li>
	</ol>
</li>
	<li>
小鼠睾丸胞浆裂解制剂的制备
	<ol>
<li>解剖小鼠睾丸。在膜中做一个小切口, 收集睾丸的精管, 并将其转移到含有5毫升 DPBS 的15毫升圆锥管内, 辅以0.1 毫米 phenylmethylsulfonyl 氟化物 (PMSF)。</li>
		<li>多次反转, 使组织剧烈混合。允许组织在冰上以单位重力沉降5分钟。</li>
		<li>除去和丢弃含有结缔组织和组织片段的多云缓冲区。重复这个过程2-3 次。其余的白色颗粒被丰富的精矿小管和生殖细胞。</li>
		<li>将精管小球转移到2毫升离心管内, 在 500 x g 处旋转1分钟。</li>
		<li>添加500µL 裂解缓冲液 (20 毫米三盐酸, pH 值 7.4, 100 毫米氯化钾, 5 毫米氯化镁2, 1 毫米, 0.5% NP-40, 1x 蛋白酶抑制剂鸡尾酒 (无 EDTA), 1 毫克/毫升肝素或200单位/毫升 RNase 抑制剂) 到小管。用吸管 triturate 组织。</li>
		<li>将悬浮液转移至小 (0.5-1.0 毫升) Dounce 均质机。用玻璃杵的七到八冲程扰乱组织。</li>
		<li>将裂解液转移到1.5 毫升离心管。</li>
		<li>离心样品在 1.2万 x g 和4°c 8 分钟, 以清除裂解。将上清液转移到新管上, 储存在冰上, 直到在蔗糖梯度上加载。</li>
		<li>收集 50 ul 的裂解液作为输入样本, 冻结它马上在-80 °c 为未来的蛋白质分析;或者在冷冻前加入 rna 纯化试剂进行 rna 分析。</li>
	</ol>
</li>
</ol>2. 蔗糖梯度的制备和离心
<ol><li>准备两个蔗糖梯度溶液 (20 毫米 HEPES, pH 值 7.4, 100 毫米氯化钾, 10 毫米氯化镁2, 1 毫米, 1x 蛋白酶抑制剂鸡尾酒), 含有10% 蔗糖或50% 蔗糖。(三盐酸, pH 值 7.4, 可以使用而不是 HEPES)。根据实验设计, 添加200个单位/毫升 RNase 抑制剂或1毫克/毫升肝素。将 ultracentrifuge 管用于 SW 41 转子进入标记块并沿该块的上层绘制线。把管子转到一个稳定的架子上。</li>
	<li>采取10毫升注射器与分层设备连接, 并填补注射器与10% 蔗糖溶液 (准备如上)。轻轻松开它在 ultracentrifuge 管的底部, 直到它到达管上的标记。</li>
	<li>用50% 蔗糖溶液填充另一注射器, 并小心地将其分层装置通过10% 蔗糖层插入管底部。从底部开始轻轻地释放蔗糖溶液, 直到它到达管子上的标记。用所提供的瓶盖封住管子。</li>
	<li>要准备蔗糖渐变, 请打开 "渐变制造器" 设备ON。使用向上或下拉按钮对板进行水平设置, 然后按完成。调平对渐变的线性度很重要。</li>
	<li>在对板进行平整后, 按研究生打开渐变菜单。转到 "渐变" 菜单上的列表, 然后选择 SW 41 Ti 转子。然后使用向上和下拉按钮从菜单列表中选择所需的蔗糖渐变。按使用。</li>
	<li>将渐变管架放在渐变制造板上。把管子转到支架上。可同时准备多达6个渐变。按运行。梯度发生器按编程速度和角度旋转管子, 形成线性梯度。准备渐变要花几分钟时间。</li>
	<li>完成过程后, 将管子放在机架中。把帽子摘下来。从 ultracentrifuge 管顶部取出与试样体积相同的体积。</li>
	<li>仔细加载 400-500 ul 的裂解液, 其中包含15-20 个在顶部的 polysomes 的260单位。把管子放在转子桶里, 平衡它们。</li>
	<li>离心机在 26万 x g 和4°c 2 小时使用 SW 41 转子。</li>
</ol>3. Polysome 分馏和样品收集
注: 虽然裂解制剂有一定的差异, 根据来源, 梯度准备和 polysome 分馏协议是相同的所有类型的裂解物。
<ol><li>离心完成后, 将转子桶与管子放在冰上。打开分数收集器和渐变分馏塔ON。单击 "分馏塔" 菜单上的扫描。把24收集管的架子放进分数收集器。</li>
	<li>用去离子水填满分馏塔一侧的冲洗池。按下十年代的冲洗键以在分馏塔上冲洗泵。将冲洗适配器与装满水的注射器连接在活塞上进行校准。</li>
	<li>打开计算机上的分馏塔软件。按校准。使用默认设置并按确定。准备从注射器中注入水。</li>
	<li>按确定进行校准。立即开始为下5秒注入水。在这段时间内, 水会流经紫外线探测器的流动细胞, 仪器将被校准。将出现符号零校准完成。该仪器已准备好进行分馏。</li>
	<li>用注射器取出冲洗适配器。将尖端连接到分馏塔的活塞上。</li>
	<li>打开黄铜空气阀, 并按空气键十年代, 以干油管和流动电池。关闭气阀。</li>
	<li>轻轻地从转子桶中取出渐变管并将其放在机架中。将管座帽涂在管子的顶部, 并小心地将管子移入管架内, 并将其锁定到位。</li>
	<li>将持有人置于分馏塔的活塞下。通常, polysomal 带可以通过眼睛看到。介绍所需的分数和体积的设置 (24 分数在 500 ul/分数通常是足够的)。适当地命名该文件。按确定, 然后转到图形按钮。在下一个窗口中, 按开始扫描。设置将显示, 请按确定。收集器将从排水沟移动到第一个分数, 活塞将进入管。当活塞到达梯度的顶端, 它会减慢到选定的速度和分数将收集。完成后, 活塞从渐变管中移出。</li>
	<li>打开黄铜气阀并按下分馏塔上的air键以检索最后一小部分。</li>
	<li>把管子从架子上移到冰上。</li>
	<li>将2卷 rna 纯化试剂添加到每一个分数, 并在液氮中快速冷冻直至 RNA 纯化。或者, 如果需要分析蛋白质, 添加乙酸酸到最终浓度为 10%, 将其浓缩为西方印迹 (见8节)。</li>
</ol>4. qPCR 数据分析中基因水平规范化的合成 RNA体外制备
注意: 本协议中使用了大肠杆菌欧帕 mRNA 进行规范化。任何其他 RNA 没有广泛的身份与研究的有机体的基因 (哺乳动物或利什曼原虫) 可以使用。
<ol><li>用含有欧帕基因30的质粒的标准 PCR 反应制备含有 SP6 启动子序列的欧帕 DNA 片段。</li>
	<li>准备100µL 的混合物:80 毫米 HEPES, pH 值 7.5, 16 毫米氯化镁2, 2 毫米胺, 10 毫米多线, 3 毫米 ATP, 3 毫米 CTP, 3 毫米 UTP, 3 毫米 GTP, 0.5 U/ul RNase 抑制剂, 1 微克欧帕 PCR DNA, 3 ul SP6 RNA 聚合酶, 0.005 U/ul 焦磷酸酶。</li>
	<li>孵育40°c 为2小时。</li>
	<li>rna 纯化试剂盒纯化 rna。</li>
	<li>用分光光度法测定浓度, 用琼脂糖凝胶电泳检查。</li>
</ol>5. RNA 分离从梯度组分和 cDNA 准备
注: 如果使用 RNase 抑制剂作为核糖核酸抑制剂, 则直接使用此协议进行 RNA 纯化。然而, 当用作核糖核酸抑制剂时, 肝素会抑制逆转录酶在 cDNA 制备中的应用。因此, 如果用肝素在溶解缓冲和梯度中使用, 则需要额外的 RNA 纯化。请参阅6节, 以制备 RNA 为 cDNA 合成如果使用肝素。
<ol><li>将含有 rna 纯化试剂的样品解冻, 加入20的合成 RNA 作为 qPCR 结果规范化的内部控制。根据制造商的协议进行 RNA 的准备, 除了一个修改。添加1µL 的 RNA 级糖原 (20 µg) 前异丙醇沉淀。将 RNA 颗粒溶于20-25 µL 的 RNase 水中。 注意: 糖原作为载体, 有助于避免损失和可视化 RNA 颗粒在纯化过程中。欧帕 mRNA 用于 qPCR 反应的进一步规范化。</li>
	<li>用分光光度法测定 RNA 浓度, 确保产量充足。将含有40S、60S 和 monosomes 的 RNA 分数的相等体积组合为 prepolysomes。含有2-4 核糖体的分数作为轻 polysomes 和5-8 核糖体的分数组合为重 polysomes。</li>
	<li>使用5-10 µL 的 RNA 从组合的分数, 以准备基因使用套件和以下制造商的建议。</li>
	<li>添加80µL 核酸酶游离水到20µL 的 cDNA。将 cDNA 样本冷冻-20 摄氏度。</li>
</ol>6. 从肝素污染中提取 RNA
注: 肝素抑制核酸处理酶 (如逆转录)。因此, 当肝素用于溶解缓冲和/或梯度时, 使用这个附加的纯化协议。
<ol><li>添加 LiCl 到1米最终浓度的纯化 RNA 样品。</li>
	<li>将样品混合在冰上孵育1小时。</li>
	<li>旋转样品在 1.6万 x g 和4°c 为15分钟。</li>
	<li>用吸管将上清液取出即可完成。</li>
	<li>空气干燥的颗粒约5分钟。</li>
	<li>在 RNase 水的初始体积内重新悬浮颗粒。</li>
	<li>执行分光光度法测量 260 nm, 以确定 RNA 的浓度。通常, 样品的损失是极小的。</li>
</ol>7. qPCR 和基因分布的数据分析
<ol><li>结合 10.2 ul 的水, 20 ul 的 SYBR 绿色, 4.8 ul 的基因特异性引物 (2.5 微米每套), 5 ul 的 cDNA, 混合良好和负载 10 ul 每井在 triplicates 成384井板块。</li>
	<li>盖板与胶膜紧密和离心板在 1800 x g 5 分钟。</li>
	<li>使用实时 PCR 仪在表 1中显示的条件下设置 qPCR 反应。</li>
	<li>使用循环阈值 (CT) 和比较 Ct (ΔΔCT) 方法31计算 prepolysomes、轻型和重 polysomes 中 mRNA 分布的百分比 (%), 如所述32与一个修改。在 qPCR 数据分析中使用合成 RNA (欧帕) 进行数据规范化。合成 RNA 提供了一个规范化的控制, 允许计算相对基因水平, 并比较在不同的梯度分数。</li>
</ol>8. 西方印迹法分析 Polysomal 馏分中的蛋白质
<ol><li>从 100% (瓦特/v) 股票, 添加乙酸酸 (TCA) 到选定的分数 (500 ul) 到最后浓度为 10%, 保持在冰上至少15分钟, 离心机在一个离心为5分钟, 放弃上清, 洗两次与冰冷的丙酮和溶解在 25 ul S用于电泳的 DS 页样本加载缓冲区。</li>
	<li>负载在 SDS 页和进行标准电泳与以下转移到 PVDF 膜。继续进行西方印迹33。</li>
</ol>

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作者没有什么可透露的。

蔗糖梯度结合 RNA 和蛋白质分析 Polysome 分馏是分析个体基因或整个 translatome 的平移状态以及调节平移的蛋白质因子作用的有力方法。机械在正常的生理或疾病状态。Polysomal 分析是一种特别适合于研究生物体中的平移调节的技术, 如 trypanosomatids 包括利什曼原虫, 其中转录控制很大程度上缺乏, 基因表达调控主要发生在翻译过程中。
在这里, 我们描述了一个 polysome 分馏协?...

细胞基因表达调控由转录、转录后水平和翻译后机制控制。在深 RNA 测序方面的进展允许在一个空前的水平上研究基因组范围内的稳态 mRNA 水平。然而, 最近的研究发现, 稳态 mRNA 水平并不总是与蛋白质生产相关1,2。一个单独的成绩单的命运是非常复杂的, 取决于许多因素, 如内部/外部刺激, 压力,等。在蛋白质合成过程中, 基因表达调控为在变化条件下快速反应提供了另一层表达控制。Polysome (或 "polyribosome") 剖面分析, 积极翻译核糖体的分离和可视化, 是研究蛋白质合成调控的有力方法。虽然, 它的第一个实验应用出现在二十世纪六十年代3中, polysome 分析目前是蛋白质翻译研究4中最重要的技术之一。单基因可由多个核糖体转化导致形成 polysome。转录可以停滞在核糖体与放线菌酮5和基因包含不同数量的 polysomes 可以在 polysome 分馏过程中分离的蔗糖梯度离心 6, 7 ,8,9. polysomal 分数的 RNA 分析然后允许测量基因组范围内单个基因的平移状态的变化, 在不同的生理条件下4,7,10. 该方法还用于揭示 5 ' UTR 和 3 ' UTR 序列在 mRNA 可译性的控制中的作用11, 检查 miRNAs 在平移抑制中的角色12, 揭开核糖体合成13 的缺陷, 并了解核糖体相关蛋白与人类疾病的作用14,15。在过去的十年中, 在翻译过程中对基因表达调控的作用越来越大, 这说明了它在人类疾病中的重要性。在癌症、代谢和神经退行性疾病中进行平移控制的证据非常惊人15,16,17,18。例如, eIF4E-dependent 平移控制的失调有助于自闭症相关的赤字15和 FMRP 参与停滞的核糖体基因链接到自闭症14。因此, polysomal 分析是研究多种人类疾病的翻译调节缺陷的重要工具。
不同生理条件下 polysomal 分数的蛋白质分析, 对翻译过程中核糖体相关因素的作用进行了剖析。polysome 分析技术已用于许多物种, 包括酵母, 哺乳动物细胞, 植物和原生动物10,19,20,21。像锥和利什曼原虫这样的原生动物寄生虫表现出有限的基因表达转录控制。他们的基因组被组织成 polycistronic 基因簇, 缺乏启动子调控的转录22。相反, 发展基因表达主要控制在蛋白质的翻译水平和 mRNA 稳定性的 trypanosomatid 物种23,24。因此, 在缺乏转录调控的情况下理解平移控制对这些生物体尤为重要。Polysomal 分析是研究利什曼原虫25、26、27、28中基因表达转录后水平调控的有力工具。
通过实时定量 PCR (RT-qPCR) 和转录的下一代测序以及蛋白质组学技术检测个体基因水平的最新进展, 将 polysomal 分析的解决方案和优势带到了一个新的层次。通过对单个 polysomal 分数的分析, 结合蛋白质组学分析的方法, 可以进一步扩大对各细胞的使用, 以监测整个基因型范围内体细胞的平移状态。这使得在不同的生理和病理条件下, 识别新的分子玩家调节翻译。在这里, 我们提出了一个通用的 polysome 分析协议, 用于三种不同的模型: 寄生虫利什曼原虫少校, 培养的人类细胞和动物组织。本文就不同生物体的细胞裂解物的制备、梯度条件的优化、RNase 抑制剂的选择、qPCR、印迹和 RNA 电泳等方面的应用提出建议, 以分析 polysome 分数。

<table>
	<tbody>
		<tr>
			<td>Instruments:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient master</td>
			<td>Biocomp Instruments Inc.</td>
			<td>108</td>
		</tr>
		<tr>
			<td>Piston Gradient Fractionator</td>
			<td>Biocomp Instruments Inc.</td>
			<td>152</td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>Gilson, Inc.</td>
			<td>FC203B</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop One</td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ts2-FL/Ts2</td>
		</tr>
		<tr>
			<td>2720 Thermal Cycler</td>
			<td>Applied Biosystems by Life Technologies</td>
			<td>4359659</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Panasonic Healthcare Co.</td>
			<td>MCO-170A1CUV</td>
		</tr>
		<tr>
			<td>HERATHERM incubator</td>
			<td>Thermo Scientific</td>
			<td>51028063</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet, class II, type A2</td>
			<td>NuAire Inc.</td>
			<td>NU-543-400</td>
		</tr>
		<tr>
			<td>Revco freezer</td>
			<td>Revco Technologies</td>
			<td>ULT1386-5-D35</td>
		</tr>
		<tr>
			<td>Beckman L8-M Ultracentifuge</td>
			<td>Beckman Coulter</td>
			<td>L8M-70</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor SW41</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
		</tr>
		<tr>
			<td>Swing-bucket rotor</td>
			<td>Eppendorf</td>
			<td>A-4-62</td>
		</tr>
		<tr>
			<td>Fixed angle rotor</td>
			<td>Eppendorf</td>
			<td>F-45-30-11</td>
		</tr>
		<tr>
			<td>Quant Studio 12K Flex Real-Time PCR machine 285880228</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4470661</td>
		</tr>
		<tr>
			<td>TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0102</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>02-671-51B</td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triax software&#160;</td>
			<td>Biocomp Instruments Inc.</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides, dual chamber for cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
		</tr>
		<tr>
			<td>Open-top polyclear centrifuge tubes, (14 mm x 89 mm)</td>
			<td>Seton Scientific</td>
			<td>7030</td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>BD</td>
			<td>309646</td>
		</tr>
		<tr>
			<td>BD Syringe 3 mL23 Gauge 1 Inch Needle</td>
			<td>BD</td>
			<td>10020439</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 14 cm</td>
			<td>Thermo Scientific</td>
			<td>168381</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 9 cm</td>
			<td>Thermo Scientific</td>
			<td>172931</td>
		</tr>
		<tr>
			<td>Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES</td>
			<td>Thermo Scientific</td>
			<td>569-0020</td>
		</tr>
		<tr>
			<td>BioLite 75 cm3 flasks</td>
			<td>Thermo Scientific</td>
			<td>130193</td>
		</tr>
		<tr>
			<td>Nunc 50 mL conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339653</td>
		</tr>
		<tr>
			<td>Chemicals:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol LS</td>
			<td>Ambion by Life Technologies</td>
			<td>10296028</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1378-5KG</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium-high glucose (DMEM)</td>
			<td>Sigma</td>
			<td>D6429-500ML</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F0926-50ML</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2x6H2O)</td>
			<td>Acros Organics</td>
			<td>AC413415000</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9541-500G</td>
		</tr>
		<tr>
			<td>Nonidet P 40 (NP-40)</td>
			<td>Fluka (Sigma-Aldrich)</td>
			<td>74385</td>
		</tr>
		<tr>
			<td>Recombinant Rnasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3993-1MU</td>
		</tr>
		<tr>
			<td>cOmplete Mini EDTA-free protease inhibitors</td>
			<td>Roche Diagnostics</td>
			<td>11836170001</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502-1L</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698-1G</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>194002</td>
		</tr>
		<tr>
			<td>Dithiotreitol (DTT)</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Fisher Scientific</td>
			<td>BP-1302-10</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>S316-212</td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Life Technologies</td>
			<td>22600050</td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma</td>
			<td>P8833-100MG</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-3KG</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma</td>
			<td>T4049-100ML</td>
		</tr>
		<tr>
			<td>Hgh Capacity cDNA Reverse Transcriptase Kit</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4368814</td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4367659</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144SI-212</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26324</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Sigma</td>
			<td>221473-500G</td>
		</tr>
		<tr>
			<td>Anti-RPL11 antibody</td>
			<td>Abcam</td>
			<td>ab79352</td>
		</tr>
		<tr>
			<td>Ribosomal protein S6 (C-8) antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-74459</td>
		</tr>
		<tr>
			<td>1xM199</td>
			<td>Sigma</td>
			<td>M0393-10X1L</td>
		</tr>
		<tr>
			<td>Lithium cloride</td>
			<td>Sigma</td>
			<td>L-9650</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D128-500</td>
		</tr>
		<tr>
			<td>Gel Loading Buffer II</td>
			<td>Thermo Scientific</td>
			<td>AM8546G</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Thermo Scientific</td>
			<td>16500-100</td>
		</tr>
		<tr>
			<td>Trichloracetic acid (TCA)</td>
			<td>Fisher Scientific</td>
			<td>A322-100</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS chemiluminescent substrate</td>
			<td>Thermo Scientific</td>
			<td>34580</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>BP531-500</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Sigma</td>
			<td>L5750-1KG</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626-5G</td>
		</tr>
		<tr>
			<td>RNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma</td>
			<td>A1852-1VL</td>
		</tr>
		<tr>
			<td>Cytosine 5&#39;-triphosphate disodium salt hydrate (CTP)</td>
			<td>Sigma</td>
			<td>C1506-250MG</td>
		</tr>
		<tr>
			<td>Uridine 5&#39;-triphosphate trisodium salt hydrate (UTP)</td>
			<td>Sigma</td>
			<td>U6625-100MG</td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate sodium salt hydrate (GTP)</td>
			<td>Sigma</td>
			<td>G8877-250MG</td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase</td>
			<td>NEB</td>
			<td>M0207S</td>
		</tr>
		<tr>
			<td>Pyrophoshatase</td>
			<td>Sigma</td>
			<td>I1643-500UN</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266-1G</td>
		</tr>
	</tbody>
</table>

polysome profiling in leishmania, human cells and mouse testis

正确的蛋白质表达在适当的时间和正确的数量是正常细胞功能和生存在一个快速变化的环境的基础。长期以来, 基因表达研究以转录水平的研究为主。然而, 基因的稳态水平与蛋白质的生产没有很好的关联, 基因的可译性因条件而变化很大。在某些有机体, 象寄生虫利什曼原虫, 蛋白质表达在翻译水平上主要被调控。最近的研究表明, 蛋白质翻译失调与癌症、新陈代谢、神经变性和其他人类疾病有关。Polysome 分析是研究蛋白质翻译规律的有力方法。它允许测量个体基因的平移状态或在全基因组范围内检查翻译。这种技术的基础是分离 polysomes, 核糖体, 他们的亚基和自由基因在离心的细胞质裂解过程中通过蔗糖梯度。在这里, 我们提出了一个通用的 polysome 分析协议, 用于三种不同的模型-寄生虫利什曼原虫少校, 培养的人类细胞和动物组织。利什曼原虫细胞在悬浮体中自由生长, 培养的人细胞生长在粘附单层, 而小鼠睾丸代表动物组织样本。因此, 该技术适用于所有这些来源。polysomal 分数分析的协议包括通过 rt-pcr 检测个体 mRNA 水平、qPCR 蛋白和用电泳分析核糖体 rna。通过对基因与核糖体在转录水平上的研究, 通过对核糖体相关蛋白的分型质谱分析, 可以进一步扩展该方法。该方法可以很容易地调整到其他生物模型。

在本研究中, 我们描述了 polysomal 分析技术在三种不同来源的应用: 寄生性的利什曼原虫少校、培养的人体细胞和小鼠睾丸。利什曼原虫细胞在悬浮液培养基中自由生长, 培养的人细胞生长在板上的贴膜上, 小鼠睾丸代表组织样本。该方法可以很容易地调整到其他类型的自由生长细胞在悬浮, 不同类型的组织, 或从另一个有机体, 和不同类型的培养细胞。该方法由...

Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

polysome 分析技术的总体目标是分析个体基因或转录基因在蛋白质合成过程中的平移活动。该方法对健康和多人疾病的蛋白质合成调控、翻译活化和抑制的研究具有重要意义。

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

polysome-profiling-in-leishmania-human-cells-and-mouse-testis

Research

JoVE Journal

Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

17.9K 次观看.  Texas Tech University Health Sciences Center. polysome 分析技术的总体目标是分析个体基因或转录基因在蛋白质合成过程中的平移活动。该方法对健康和多人疾病的蛋白质合成调控、翻译活化和抑制的研究具有重要意义。

视频: Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

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 ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

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 ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.