Name: phosphoproteomic strategy for profiling osmotic stress signaling in arabidopsis
Uploaded: 2020-06-25T14:00:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis at JoVE.com

这项工作得到了中国科学院战略优先研究项目格兰特XDB27040106的支持。

1. 样品准备
<ol><li>在铝箔中收获控制和应力处理的幼苗（1克），并闪过在液氮中冷冻样品。 注意:通常从两周大的幼苗中观察到比成熟植物高的蛋白质浓度。一克幼苗产生大约10毫克的蛋白质解析，这足以进行MS分析。所有离心步骤在第 1 步中均在 16，000 x g 下进行。</li>
	<li>用砂浆和充满液氮的害虫将冷冻幼苗磨成细粉。</li>
	<li>添加1 mL的裂解缓冲区（（6 M瓜尼丁-HCl在100 mM特里斯-HCl， pH 8.5） 与 10 mM 三叶草 （2-碳甲基） 盐酸磷化物 （TCEP）， 40 mM 2-氯乙酰胺 （CAA）， 蛋白酶抑制剂和磷酸酶抑制剂鸡尾酒） 到砂浆和混合与害虫的帮助.</li>
	<li>将植物裂解到 1.5 mL 管中，在 95 °C 下加热 5 分钟。</li>
	<li>将管子放在冰上10分钟。在冰上松下管子10s，暂停10s，重复三次。</li>
	<li>将管子离心 20 分钟，将 150μL 的植物解压成 1.5 mL 管。</li>
	<li>将 600 μL 的 100% 甲醇加入管中。漩涡和旋转管。</li>
	<li>在管子中加入 150 μL 的 100% 氯仿。漩涡和旋转管。</li>
	<li>将 450μL 的 ddH2O 添加到管中。涡流和离心管3分钟。</li>
	<li>丢弃上一层。将 600 μL 的 100% 甲醇加入管中。将管子离心3分钟，然后丢弃溶液。</li>
	<li>用 600 μL 的 100% 甲醇清洗蛋白质颗粒。丢弃溶液，使蛋白质颗粒干燥。</li>
	<li>将蛋白质颗粒重新悬生到 600μL 的消化缓冲区（12 mM 脱氧糖酸钠 （SDC）/12 mM 酸酸钠 （SLS）， 在 100 m Tris-HCl， pH 8.5） 中。将管子声波化，直到悬架均匀化。</li>
	<li>使用 BCA 套件测量蛋白质浓度，并使用消化缓冲器将浓度调整到 4 μg/μL。将 100 μL 的利酸盐（400μg 蛋白质）转移到新管中。</li>
	<li>在管子中加入 292 μL 的 50 m m 三乙酸氢铵 （TEAB） 和 8 μL 的 Lys-C （2.5 单位/mL）。在37°C下孵育管子3小时。</li>
	<li>在管子中加入 100μL 的 50 mm TEAB 和 8μg 试管。在37°C下孵育管12小时。</li>
	<li>在管子中加入 25 μL 的 10% 三氟乙酸 （TFA）。涡流和离心管在4°C下20分钟。</li>
	<li>将超盐剂转移到有条件的脱盐柱中进行脱盐。使用真空离心机集中器干燥流体。 注:用1 mL甲醇激活树脂，然后在80%丙烯酸酯（ACN）中激活1 mL的0.1%TFA。在 5% ACN 中用至少 3 mL 的 0.1% TFA 洗掉有机溶剂。将第 1.16 步准备的酸化肽样品装入柱中，然后用至少 3 mL 的 0.1% TFA 在 5% ACN 中清洗柱子。高盐浓度样品可能需要更多洗涤。在 80% ACN 中，用 1 mL 的 0.1% TFA 排出磷肽。</li>
</ol>2. 串联质量标签 （TMT） 标签
<ol><li>将干燥的肽重新悬生成 100 μL 的 200 mM 4-（2-羟基）-1-管道氨酸（HEPES），pH 8.5。</li>
	<li>在 40 μL 的无水性 ACN 中溶解每个通道的 TMT 试剂 （0.8 毫克）。涡流TMT试剂管5分钟，并旋转下来。</li>
	<li>将 40 μL TMT 转移到样品管中，并在室温下孵育 1 小时。 注:在此实验中，通道 126 到 128 被标记为三种未经曼尼托处理的植物的生物复制品，而通道 129 到 131 则标有三种未经曼尼托处理的植物的生物复制品。</li>
	<li>加入8 μL的5%羟基胺，孵育15分钟。</li>
	<li>在 5 mL 管中混合 6 个样本，并添加 14 μL 的 10% TFA。</li>
	<li>在管子中添加 3，876 μL 的 0.1% TFA。漩涡和旋转下来。</li>
	<li>将溶液转移到有条件的脱盐柱中进行脱盐。使用蒸发器干燥蒸发器。</li>
</ol>3. 准备 IMAC 舞台提示
<ol><li>使用 16 G 钝端针穿透聚丙烯褶皱盘。 注意:将切割机垂直于磁盘表面，并滚动切割机几次，以确保磁盘完全切除。将磁盘放入培养皿中存储。将针头放入 200 μL 移液器尖端，然后使用柱塞将毛刺推入尖端。</li>
	<li>使用柱塞将褶皱轻轻压入尖端，以盖住尖端。<ol>
<li>将炸盘放在具有相同压力的提示中，从而提供更好的可重复性。舞台尖端生产的可重复性对于恒定的背压非常重要，这会影响离心步骤的时机和技术复制之间的可重复性。</li>
		<li>如果舞台提示在调理步骤中显示高压，则丢弃提示以避免在加载样品时潜在的提示堵塞。可能是磁盘被压得太紧了。</li>
	</ol>
</li>
	<li>倒置 Ni-NTA 旋转柱，放在 1.5 mL 管上。</li>
	<li>使用柱塞轻轻按下 Ni-NTA 珠子的褶皱，并将珠子推入管子中。 注意:请确保轻轻推动碎屑，以避免旋转柱上潜在的珠子丢失或吸附。</li>
</ol>4. Hp-RP 阶段提示的准备
<ol><li>使用 C8 磁盘为 IMAC 舞台提示准备 Hp-RP 阶段提示。 注意:不要在磁盘上施加大力，因为它可能导致密集包装的褶皱，并增加舞台尖端的后压。所有离心步骤在第 4 步中以 1，000 x g 的速度进行 5 分钟。</li>
	<li>在 100 μL 100% 甲醇中暂停 1 毫克 C18 珠，并通过尖端传递珠子溶液。</li>
	<li>将 20 μL 的缓冲区 8 （80% ACN/20% 200 mM NH4HCO2，pH 10.0） 添加到舞台尖端，并通过离心通过尖端通过缓冲器 8。</li>
	<li>在舞台尖端添加 20 μL 的缓冲区 A （200 mM NH4HCO2），并通过离心通过尖端传递缓冲 A 。 注意:在使用过程中平衡离心机，并确保 Hp-RP 阶段尖端不会完全干燥。</li>
</ol>5. 旋转适配器的准备
<ol><li>使用锋利的末端钳子刺穿 1.5 mL 管盖中央的孔。</li>
	<li>将 IMAC 舞台提示或 Hp-RP 舞台提示插入自旋适配器的孔中。 注意:确保舞台提示不靠近自旋适配器的底部。</li>
</ol>6. 使用 IMAC 阶段提示富集磷肽
<ol><li>暂停 10 毫克 Ni-NTA 珠子与 400μL 的加载缓冲区 （6% 醋酸 （AA）， pH 3.0）， 并将所有珠子溶液加载到每个尖端。通过离心将解决方案通过尖端传递。 注:除步骤6.8外，所有离心步骤均在200 x g 下进行，第6步为3分钟。</li>
	<li>装载 100 μL 的 50 mm 乙烯二胺三乙酸 （EDTA）， 从 IMAC 阶段尖端剥离镍离子，并通过离心将溶液通过尖端传递。</li>
	<li>将 100 μL 的装载缓冲器加载到舞台尖端，并通过离心将解决方案通过尖端传递。</li>
	<li>将 50 mM FeCl3 中的 100 μL（6%） 加载到舞台尖端，并通过离心将解决方案传递给尖端。Fe3+ 离子将串起到 IMAC 舞台提示。</li>
	<li>加载 100 μL 的装载缓冲器，以调节阶段尖端，并通过离心将解决方案通过尖端传递。</li>
	<li>加载 100 μL 的装载缓冲区，并准备了第 2.7 步的样品肽到阶段尖端，并通过离心将解决方案通过尖端传递。 注意:以新管子作为自旋适配器，收集样品的流经，并将流经储存在冰柜中，供将来分析。</li>
	<li>将 100 μL 的洗涤缓冲器（4.5% AA 和 25% ACN）加载到舞台尖端，并通过离心将解决方案通过尖端传递。</li>
	<li>用加载缓冲器执行第二次清洗。将 100 μL 的装载缓冲器加载到舞台尖端，然后使用 1000 × g 离心机将解决方案传递至端端 3 分钟。 注意:确保洗涤缓冲区被舞台尖端的加载缓冲器所取代，以消除排洗步骤期间磷肽的损失。在磷肽排出之前平衡 IMAC 阶段尖端，以确保 ACN 浓度低于 5%。</li>
	<li>用剪刀修剪 IMAC 舞台尖端，并将修剪过的 IMAC 舞台提示放在 Hp-RP 尖端内。确保两层舞台提示不接触离心机的盖子。</li>
</ol>7. 磷肽分馏使用 Hp-RP C18 阶段提示
<ol><li>将 100 μL 的叶芽缓冲液（200 mM 磷酸铵 （NH4H2PO4）添加到修剪过的 IMAC 阶段端，并通过离心的两层阶段提示传递解决方案。 注意:如果解决方案没有通过阶段提示，则加快旋转速度。所有离心步骤在第 7 步中以 1，000 x g 的速度进行 5 分钟。所有 Hp-RP 缓冲区都列在 表 1中。</li>
	<li>使用钳子丢弃 IMAC 舞台提示，并将 20 μL 的缓冲区 A 添加到 Hp-RP 舞台提示中，并通过离心通过尖端通过缓冲区。 注意:在丢弃之前，确保所有自助缓冲区通过 IMAC 阶段提示。</li>
	<li>添加缓冲区 1 的 20 μL 以分馏第 1 部分，并通过离心在新管中收集流出。用缓冲区 2、3、4、5、6、7 和 8 重复该过程，以在新管中收集每个分数。使用真空集中器干燥每个分数的最后一个 eluate。 注意:准备新的分馏缓冲区。以 8 根新管子作为每个分数的自旋适配器。在不同的自旋适配器中收集每个分数的分量。磷肽在基本条件下不稳定，因此由集中器干燥，或立即酸化10%TFA。</li>
</ol>8. LC-MS/MS 分析和数据分析
<ol><li>加入 5 μL 的 0.1% 的氨酸 （FA），并通过质谱仪分析样品。</li>
	<li>运行一个90分钟的梯度，每个分数有6-30%的缓冲B（80%ACN和0.1%FA）。</li>
	<li>使用高碰撞能量分离 （HCD） 将前 10 种标记的肽碎片。完成数据库搜索以识别磷化站点。</li>
	<li>将原始文件加载到 MaxQuant 软件中，命名实验，并设置分数和 PTM。 注:MaxQuant 软件的教程可在 https://www.maxquant.org/ 找到。</li>
	<li>选择记者离子 MS2 在 LC-MS/MS 运行类型和 6 倍 TMT 作为同位素标签。通过 PIF 功能启用过滤器，并将最小 PIF 设置为 0.75。</li>
	<li>选择乙酰（蛋白质N术语）、氧化（M）和磷（STY）到可变修改面板。选择卡巴米多美乙基 （C） 作为固定修改。</li>
	<li>将消化模式设置为特定，选择 Trypsin / P 作为消化酶，以及两个错过的。</li>
	<li>将 PSM 错误发现率 （FDR） 和蛋白质 FDR 设置为 0.01。将改性肽的最低分数设置为 40。</li>
	<li>将 阿拉伯塔利亚纳 数据库添加到快速文件面板中，并运行数据库搜索。</li>
	<li>完成搜索后，将磷（STY）网站的txt文件加载到波斯软件。 注:Perseus 软件的详细教程可在 https://www.maxquant.org/perseus/ 找到。</li>
	<li>过滤掉反向磷化位点。使用本地化概率 75% 作为截止点过滤掉未本地化磷化站点。</li>
	<li>使用磷化位点的强度进行统计分析。</li>
</ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+high-pH+reverse+phase+stageTip+for+sensitive+small-scale+membrane+proteomic+profiling.">Rapid high-pH reverse phase stageTip for sensitive small-scale membrane proteomic profiling.</a> Analytical Chemistry. 87 (24), 12016-12023 (2015).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+regulation+of+the+TOR+Kinase+and+ABA+receptor+balances+plant+growth+and+stress+response.">Reciprocal regulation of the TOR Kinase and ABA receptor balances plant growth and stress response.</a> Molecular Cell. 69 (1), 100-112 (2018).</li><li>Lin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+RAF-SnRK2+kinase+cascade+mediates+early+osmotic+stress+signaling+in+higher+plants.">A RAF-SnRK2 kinase cascade mediates early osmotic stress signaling in higher plants.</a> Nature Communications. 11 (1), 613 (2020).</li><li>Humphrey, S. J., Azimifar, S. B., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+phosphoproteomics+reveals+in+vivo+insulin+signaling+dynamics.">High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics.</a> Nature Biotechnology. 33 (9), 990-995 (2015).</li><li>Bekker-Jensen, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+shotgun+strategy+for+the+rapid+generation+of+comprehensive+human+proteomes.">An optimized shotgun strategy for the rapid generation of comprehensive human proteomes.</a> Cell Systems. 4 (6), 587-599 (2017).</li><li>Possemato, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+phosphoproteomic+profiling+using+titanium+dioxide+and+immunoaffinity+enrichments+reveals+complementary+phosphorylation+events.">Multiplexed phosphoproteomic profiling using titanium dioxide and immunoaffinity enrichments reveals complementary phosphorylation events.</a> Journal Proteome Research. 16 (4), 1506-1514 (2017).</li><li>Hogrebe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+common+quantification+strategies+for+large-scale+phosphoproteomics.">Benchmarking common quantification strategies for large-scale phosphoproteomics.</a> Nature Communications. 9 (1), 1045 (2018).</li><li>Wong, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteomics+of+Arabidopsis+Highly+ABA-Induced1+identifies+AT-Hook-Like10+phosphorylation+required+for+stress+growth+regulation.">Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook-Like10 phosphorylation required for stress growth regulation.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (6), 2354-2363 (2019).</li><li>Yang, F., Melo-Braga, M. N., Larsen, M. R., Jorgensen, H. J., Battle Palmisano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=through+signaling+between+wheat+and+the+fungal+pathogen+Septoria+tritici+revealed+by+proteomics+and+phosphoproteomics.">through signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics.</a> Molecular &amp; Cell Proteomics. 12 (9), 2497-2508 (2013).</li><li>Tsai, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+Mass+Tag+labeling+facilitates+reversed-phase+liquid+chromatography-mass+spectrometry+analysis+of+hydrophilic+phosphopeptides.">Tandem Mass Tag labeling facilitates reversed-phase liquid chromatography-mass spectrometry analysis of hydrophilic phosphopeptides.</a> Analytical Chemistry. 91 (18), 11606-11613 (2019).</li></ol>

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植物蛋白质组和磷蛋白质组的动态范围和复杂性仍然是磷蛋白分析深度的限制因素。尽管单运行LC-MS/MS分析能够识别10，000个磷化位点21，22，但整个植物磷蛋白的覆盖率仍然有限。因此，在分析植物信号网络应对环境压力的全球视图时，需要提供高灵敏度和卓越分离效率的磷蛋白工作流。商用高压液体色谱（HPLC）柱基色谱是MS分析23、24</sup...

高盐度、低温和干旱引起渗透应力，这是影响植物生产力的主要环境因素。蛋白质磷化是植物对渗透应激3、4、5的反应中最重要的转化后修饰之一。SNF1相关的蛋白激酶2s（SnRK2s）参与渗透应激信号6。SnRK2家族的10名成员中有9人因对渗透性压力7、8表现出显著的激活作用。snrk2.1/2/3/4/5/6/7/8/9/10突变（snrk2-dec）突变在所有 10 SnRK2 显示对渗透应激的超敏性。在snrk2-dec突变体中，渗透应激引起的不溶胶1，4，5-三磷酸盐（IP3）、苦酸（ABA）生物合成和基因表达的积累被强烈减少，突出了SnRK2s在渗透应激反应中的重要作用。然而，目前还不清楚SnRK2s激酶是如何调节这些生物过程的。分析针对渗透应激的磷蛋白变化是弥合这一差距和描绘植物中渗透应激触发防御机制的有效方法。
质谱学（MS）是测绘植物磷蛋白9的一项强有力的技术。然而，由于植物蛋白质组的动态范围和植物草酸盐4的复杂性，植物磷蛋白的定性仍然是一个挑战。为了克服这些挑战，我们开发了一种通用的植物磷蛋白工作流程，它消除了光合作用颜料和次要代谢物等不必要的干扰，并实现了植物磷蛋白10的深度覆盖。在MS分析11、12、13、14、15、16之前，已经开发出几种磷肽浓缩方法，如固定金属离子亲和度色谱（IMAC）和金属氧化物色谱（MOC）。酸性非磷肽与磷肽共同净化是磷肽检测的主要干扰物。此前，我们标准化了 IMAC 装载缓冲区的 pH 值和有机酸浓度，以消除非磷肽的结合，获得 90% 以上的浓缩特异性，绕过了分馏前步骤11。
磷肽富集和分馏多步骤过程中的样品流失妨碍了磷肽鉴定的敏感性和磷蛋白覆盖深度。停止和去提取技巧（阶段提示）是移液器提示，包含小磁盘，以盖住尖端的末端，它可以与色谱相结合的肽分馏和清洁17。避免样品在管子之间转移，可以最大限度地减少阶段提示过程中的样品损失。我们已经成功地在Ga3+-IMAC和Fe3+-IMAC中实现了阶段提示，将低丰富的多磷酸肽与单磷酸肽分离出来，从而提高了人类磷蛋白15的深度。此外，与强 cation 交换 （SCX） 和强阴离子交换 （SAX） 色谱18相比，高 pH 反相 （Hp-RP） 阶段尖端的使用表明人类膜蛋白质组的覆盖范围更广。因此，集成 IMAC 和 Hp-RP 阶段尖端技术可以提高植物磷蛋白的覆盖率，具有简单性、高特异性和高吞吐量。我们已经证明，这一战略从 阿拉比多普西 斯幼苗中确定了2万多个磷化位点，代表了植物磷蛋白体19的深度。
在这里，我们报告了阿拉伯磷蛋白分析阶段基于尖端的磷蛋白化方案 。 此工作流程用于研究野生型和 snrk2-dec 突变幼苗的磷蛋白扰动，以应对渗透应激。磷蛋白分析显示，磷化位点与激酶活化和早期渗透应激信号有关。对野生型和 snrk2-dec 突变磷蛋白组数据的比较分析，发现了一种类似Raf的激酶（RAF）-SnRK2激酶级联，在高植物的奥斯莫尔应激信号中起着关键作用。

<table><tbody><tr><td>1.5 mL tube</td><td>eppendorf</td><td>22431081</td><td>Protein LoBind, 1.5 mL, PCR clean, colorless, 100 tubes</td></tr><tr><td>200 &amp;micro;L pipet tip</td><td>Gilson</td><td>F1739311</td><td></td></tr><tr><td>2-chloroacetamide</td><td>Sigma-Aldrich</td><td>C0267</td><td></td></tr><tr><td>acetic acid</td><td>Sigma-Aldrich</td><td>5438080100</td><td></td></tr><tr><td>acetonitrile</td><td>Sigma-Aldrich</td><td>271004</td><td></td></tr><tr><td>ammonium hydroxide</td><td>Sigma-Aldrich</td><td>338818</td><td></td></tr><tr><td>ammonium phosphate monbasic</td><td>Sigma-Aldrich</td><td>216003</td><td></td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>blunt-ended needle</td><td>Hamilton</td><td>90516</td><td>Kel-F hub (KF), point style 3, gauge 16</td></tr><tr><td>C18-AQ beads</td><td>Dr. Maisch</td><td>ReproSil-Pur-C18-AQ 5 &amp;micro;m</td><td></td></tr><tr><td>C8 Empore disk</td><td>3 M</td><td>2214</td><td>47 mm</td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>22620444</td><td></td></tr><tr><td>chloroform</td><td>Sigma-Aldrich</td><td>CX1058</td><td></td></tr><tr><td>data analysis software</td><td></td><td>Perseus 1.6.2.1</td><td>https://maxquant.net/perseus/</td></tr><tr><td>ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>formic acid</td><td>Sigma-Aldrich</td><td>5330020050</td><td></td></tr><tr><td>Frits</td><td>Agilent</td><td>12131024</td><td>Frits for SPE Cartridges</td></tr><tr><td>Guanidine hydrochloride</td><td>Sigma-Aldrich</td><td>50933</td><td></td></tr><tr><td>H2O</td><td>Sigma-Aldrich</td><td>1153334000</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Iron (III) chloride</td><td>Sigma-Aldrich</td><td>157740</td><td></td></tr><tr><td>LTQ-orbitrap</td><td>Thermo Fisher Scientific</td><td>Velos Pro</td><td></td></tr><tr><td>mass spectrometer</td><td>Thermo Fisher Scientific</td><td>LTQ-Orbitrap Velos Pro</td><td></td></tr><tr><td>methanol</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>nano LC</td><td>Thermo Fisher Scientific</td><td>Easy-nLC 1000</td><td></td></tr><tr><td>Ni-NTA spin column</td><td>Qiagen</td><td>31014</td><td></td></tr><tr><td>N-Lauroylsarcosine sodium salt</td><td>Sigma-Aldrich</td><td>L9150</td><td></td></tr><tr><td>plunger</td><td>Hamilton</td><td>1122-01</td><td>Plunger assembly N, RN, LT, LTN for model 1702 (25 &amp;mu;l)</td></tr><tr><td>search engine software</td><td></td><td>MaxQuant 1.5.4.1</td><td>&lt;u&gt;https://www.maxquant.org&lt;/u&gt;</td></tr><tr><td>SEP-PAK Cartridge 50 mg</td><td>Waters</td><td>WAT054960</td><td></td></tr><tr><td>sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>SpeedVac</td><td>Thermo Fisher Scientific</td><td>SPD121P</td><td></td></tr><tr><td>TMT 6-plex</td><td>Thermo Fisher Scientific</td><td>90061</td><td></td></tr><tr><td>Triethylammonium bicarbonate buffer</td><td>Sigma-Aldrich</td><td>T7408</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>91707</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Trizma hydrochloride</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr></tbody></table>

phosphoproteomic strategy for profiling osmotic stress signaling in arabidopsis

蛋白质磷化对于控制渗透条件下的酶活性和基因表达至关重要。基于质谱（MS）的磷蛋白经济学改变了研究植物信号转导的方法。然而，需要大量的起始材料和更长的MS测量时间来达到覆盖深度，一直是全球植物磷蛋白变化高通量研究的限制因素。为了提高植物磷蛋白的灵敏度和通量，我们开发了一种停止和去提取（阶段）尖端的磷蛋白经济学方法，并结合Tandem质量标签（TMT）标签，对植物磷酸化扰动进行快速、全面的分析，以应对渗透应激。利用阶段尖端技术的简单性和高通量，整个过程大约需要一个小时，使用两个提示完成磷肽的浓缩、分馏和样品清洁步骤，表明该方法易于使用且效率高。这种方法不仅提供了深入的植物磷蛋白学分析（+11，000 磷肽识别），而且还显示了相邻分数之间的卓越分离效率（+lt; 5%重叠）。此外，还利用TMT标签实现了多路复用，以量化野生型和 snrk2 脱钩突变植物的磷蛋白变化。这种方法已成功地用于揭示拉夫样激酶的磷化事件，以回应渗透应激，这揭示了对陆地植物早期渗透信号的理解。

为了演示此工作流程的性能，我们利用 IMAC 阶段提示加上 Hp-RP 阶段尖端分馏来测量野生型和 snrk2-dec 突变幼苗中具有或无需曼尼托治疗的磷蛋白变化 30 分钟。每个样本都是在生物三元体中进行的，实验工作流程以 图1表示。每个样品的消化肽（400微克）被标记为一个TMT-6plex通道，汇总和脱盐。磷肽使用 IMAC 阶段尖端进一步丰富，随后 Hp-RP 阶段尖端将纯化磷肽分成八个...

Watch this Scientific Journal Video about Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis at JoVE.com

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

这里介绍的是一种磷蛋白法，即停止和去提取基于磷蛋白的尖端，它提供了高通量和深度覆盖 的阿拉比多普西 磷蛋白。这种方法概述了阿拉伯语中渗透应激信号 的概述。

分析阿拉伯恐惧症中渗透应激信号的磷蛋白分析策略

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

phosphoproteomic-strategy-for-profiling-osmotic-stress-signaling

Research

JoVE Journal

Biochemistry

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

5.1K 次观看.  Carnegie Institute for Science. 这里介绍的是一种磷蛋白法，即停止和去提取基于磷蛋白的尖端，它提供了高通量和深度覆盖 的阿拉比多普西 磷蛋白。这种方法概述了阿拉伯语中渗透应激信号 的概述。

视频: Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

科研

生物化学

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

环境科学

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

发育生物学

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

免疫与感染

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

生物学

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

测定蛋白酶体降解的植物无细胞系统

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with 35S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.

分析蛋白导入进强调，从植物中分离叶绿体

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

通过表皮表型评分 鉴定 参与气孔发育的基因

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

气候相关胁迫下的马铃薯恢复力分析

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal&#160;the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

作者聚焦:使用 PlantScreen 机器人平台揭示植物对非生物胁迫的响应

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

基于图像的高通量表型分析用于确定马铃薯对单一胁迫和联合胁迫的形态和生理响应

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...

分析阿拉伯恐惧症中渗透应激信号的磷蛋白分析策略

摘要

探索更多视频

此视频中的章节

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

代谢标记和膜分馏的比较蛋白质组学分析

植物蛋白复合体的翻译后修饰的鉴定

测定蛋白酶体降解的植物无细胞系统

分析蛋白导入进强调，从植物中分离叶绿体

通过表皮表型评分鉴定参与气孔发育的基因

基于图像的高通量表型分析用于确定马铃薯对单一胁迫和联合胁迫的形态和生理响应

分析阿拉伯恐惧症中渗透应激信号的磷蛋白分析策略

摘要

探索更多视频

此视频中的章节

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

代谢标记和膜分馏的比较蛋白质组学分析

植物蛋白复合体的翻译后修饰的鉴定

测定蛋白酶体降解的植物无细胞系统

分析蛋白导入进强调，从植物中分离叶绿体

通过表皮表型评分 鉴定 参与气孔发育的基因

基于图像的高通量表型分析用于确定马铃薯对单一胁迫和联合胁迫的形态和生理响应

通过表皮表型评分鉴定参与气孔发育的基因