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>

<ol>
	<li>Hasegawa, P. M., Bressan, R. A., Zhu, J. K., Bohnert, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+cellular+and+molecular+responses+to+high+salinity.">Plant cellular and molecular responses to high salinity.</a> Annual Review Plant Physiology Plant Molecualr Biology. 51, 463-499 (2000).</li><li>Janmohammadi, M., Zolla, L., Rinalducci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+temperature+tolerance+in+plants:+Changes+at+the+protein+level.">Low temperature tolerance in plants: Changes at the protein level.</a> Phytochemistry. 117, 76-89 (2015).</li><li>Umezawa, T., Takahashi, F., Shinozaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+networks+in+the+abscisic+acid+signaling+pathway.">Phosphorylation networks in the abscisic acid signaling pathway.</a> Enzymes. 35, 27-56 (2014).</li><li>Silva-Sanchez, C., Li, H., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+and+challenges+in+plant+phosphoproteomics.">Recent advances and challenges in plant phosphoproteomics.</a> Proteomics. 15 (5-6), 1127-1141 (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=Mapping+proteome-wide+targets+of+protein+kinases+in+plant+stress+responses.">Mapping proteome-wide targets of protein kinases in plant stress responses.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (6), 3270-3280 (2020).</li><li>Fujii, H., Verslues, P. E., Zhu, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+decuple+mutant+reveals+the+importance+of+SnRK2+kinases+in+osmotic+stress+responses+in+vivo.">Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (4), 1717-1722 (2011).</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=Quantitative+phosphoproteomics+identifies+SnRK2+protein+kinase+substrates+and+reveals+the+effectors+of+abscisic+acid+action.">Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (27), 11205-11210 (2013).</li><li>Boudsocq, M., Barbier-Brygoo, H., Lauriere, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+nine+sucrose+nonfermenting+1-related+protein+kinases+2+activated+by+hyperosmotic+and+saline+stresses+in+Arabidopsis+thaliana.">Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana.</a> Journal of Biological Chemistry. 279 (40), 41758-41766 (2004).</li><li>Li, J., Silva-Sanchez, C., Zhang, T., Chen, S., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteomics+technologies+and+applications+in+plant+biology+research.">Phosphoproteomics technologies and applications in plant biology research.</a> Frontiers in Plant Science. 6, 430 (2015).</li><li>Hsu, C. C., 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=Universal+plant+phosphoproteomics+workflow+and+its+application+to+Tomato+signaling+in+response+to+cold+stress.">Universal plant phosphoproteomics workflow and its application to Tomato signaling in response to cold stress.</a> Molecular &amp; Cell Proteomics. 17 (10), 2068 (2018).</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=Immobilized+metal+affinity+chromatography+revisited:+pH/acid+control+toward+high+selectivity+in+phosphoproteomics.">Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics.</a> Journal of Proteome Research. 7 (9), 4058-4069 (2008).</li><li>Larsen, M. R., Thingholm, T. E., Jensen, O. N., Roepstorff, P., Jorgensen, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+enrichment+of+phosphorylated+peptides+from+peptide+mixtures+using+titanium+dioxide+microcolumns.">Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.</a> Molecular &amp; Cell Proteomics. 4 (7), 873-886 (2005).</li><li>Ruprecht, 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=Comprehensive+and+reproducible+phosphopeptide+enrichment+using+iron+immobilized+metal+ion+affinity+chromatography+(Fe-IMAC)+columns.">Comprehensive and reproducible phosphopeptide enrichment using iron immobilized metal ion affinity chromatography (Fe-IMAC) columns.</a> Molecular &amp; Cell Proteomics. 14 (1), 205-215 (2015).</li><li>Sugiyama, N., 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=Phosphopeptide+enrichment+by+aliphatic+hydroxy+acid-modified+metal+oxide+chromatography+for+nano-LC-MS/MS+in+proteomics+applications.">Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications.</a> Molecular &amp; Cell Proteomics. 6 (6), 1103-1109 (2007).</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=Sequential+phosphoproteomic+enrichment+through+complementary+metal-directed+immobilized+metal+ion+affinity+chromatography.">Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography.</a> Analytical Chemistry. 86 (1), 685-693 (2014).</li><li>Zhou, H., 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=Enhancing+the+identification+of+phosphopeptides+from+putative+basophilic+kinase+substrates+using+Ti+(IV)+based+IMAC+enrichment.">Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment.</a> Molecular &amp; Cell Proteomics. 10 (10), (2011).</li><li>Rappsilber, J., Mann, M., Ishihama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+micro-purification,+enrichment,+pre-fractionation+and+storage+of+peptides+for+proteomics+using+StageTips.">Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.</a> Nature Protocols. 2 (8), 1896-1906 (2007).</li><li>Dimayacyac-Esleta, B. 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>

作者声明没有利益冲突。

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之前降低肽复杂性的常用方法。然而，乏味的样品收集步骤和大的流出量是基于HDLC的方法在灵敏度和吞吐量方面的主要挑战。阶段尖端是执行肽分馏前或浓缩的高灵敏度和高通量工作的替代方法。这种新的磷蛋白工作流程利用异丙酸标签加上磷肽的富集和分馏，提供了更好的覆盖和深度的植物磷蛋白经济学比其他磷蛋白的工作流程25，26。
样品的pH值是决定磷肽富集特性的关键因素。pH 值可能受样品准备前一步的影响，因此在样品加载前必须检查 pH 值。如果 pH 移位，在样品中加入醋酸或氢氧化钠，将 pH 调整为 3.0。我们执行此协议，同时将磷肽排出并加载到 C18 阶段尖端。因此，在磷肽排出之前平衡 IMAC 阶段尖端非常重要，以确保 ACN 的浓度低于 5%。
分数数取决于复杂性和样本大小。通常，5 到 8 个分数足以在植物中提供磷肽识别的广泛覆盖。ACN在分馏缓冲区中的浓度可根据样品的疏水性进行调整。大多数磷肽使用 5% 到 25% ACN 浓度的范围从 C18 珠中分离出。由于磷酸盐组的额外负电荷，与非磷酸肽相比，磷肽通常更亲水。然而，在肽的N终点站上标记TMT试剂会导致标记肽27的疏水性增加，这或许可以解释为什么在我们病例的前两个部分（图2A）中识别的磷肽较少。因此，使用 TMT-零试剂和样品的方位引用的测试可用于评估每个分馏缓冲区中的分数数和 ACN 浓度。优化的缓冲区可提高TMT-6倍标记磷肽的分离效率。
基于阶段提示的分数的局限性是分数数和提示容量。由于不连续的梯度缓冲器用于阶段尖端分馏，因此很难扩展阶段尖端分离中的分数数。另一方面，连续梯度可以应用于 HPLC 列，以实现高分数数。提示容量是限制阶段提示应用的另一个因素。对于大型磷蛋白学分析（+10毫克蛋白质），最好使用基于HPLC的浓缩物，更长的柱长与更多的C18珠和分馏，这超出了阶段提示的分析规模。综合起来，阶段性尖端磷肽的富集和分馏是中小型磷蛋白分析的有用方法。此方法可与异体标记集成，实现多路复用量化。研究结果还表明，它可用于深入植物磷蛋白分析和量化。此工作流适用于其他物种和不同组织类型，用于划定专用信号网络。

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 &#181;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 &#181;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 &#956;l)</td>
		</tr>
		<tr>
			<td>search engine software</td>
			<td></td>
			<td>MaxQuant 1.5.4.1</td>
			<td>https://www.maxquant.org</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部分。每个分数都通过 90 分钟的 LC 渐变分析进行分析。原始文件是使用搜索引擎对 阿拉伯塔利亚纳 数据库进行搜索的。
共鉴定出11，077种独特的磷肽，相当于3，630种磷蛋白，其中6，852个局部磷化位点（I类，本地化概率=0.75），表明 阿拉伯磷 蛋白蛋白的覆盖面很广。分别从野生型和 snrk2-dec 突变样本中鉴定出8，107和7，248种磷肽。这说明了工作流程在为描述阿拉伯地区信号转导的全球视野提供深入报道方面 所起的效率。我们比较了8个分数中识别出的磷肽的数量。在前两个部分（第 1 和第 2 个分数）中发现了一些磷肽。然而，大多数磷肽均匀分布在其余6个部分（图2A），这表明这种方法提供了将复合磷肽与植物磷蛋白分离的能力。为了进一步证明此工作流程的分离效率，我们评估了两个相邻分数（eq. F1 到 F2）之间的磷肽重叠情况。相邻分数中磷肽重叠率不到 5%，表明 Hp-RP 阶段尖端 （图 2B）的分馏效率强劲。
利用工作流获得的数据，我们比较了在曼尼托治疗时野生型和snrk2-dec突变体的磷蛋白谱。野生型和snrk2-dec突变样本中的曼尼托治疗后，共增加了433个和380个磷化位点（图3）。其中，312种磷酸盐在野生类型中表现出诱导（FDR < 0.01），但在snrk2-dec突变植物中没有。基因本体论（GO）分析表明，磷酸化和蛋白激酶活化、磷酸盐代谢过程调节、信号转导等功能在SnRK2依赖性磷蛋白中显著丰富。有趣的是，与根发育相关的GO术语在SnRK2依赖组中也得到了丰富，与渗透应激6下根生长迟缓的表型一致。我们还在野生型和snrk2-dec突变体中发现了116种由曼尼托治疗调节的磷酸盐。这些磷蛋白独立于SnRK2，或在SnRK2激活前调解渗透应激触发信号的候选人。我们观察到，一些B4子组RAF，如RAF18（AT1G16270）、RAF24（AT2G35050）和RAF42（AT3G46920），受到渗透应力的显著提升。进一步研究表明，皇家空军激酶被渗透应激迅速激活，需要磷化和激活SnRK2s20。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61489/61489fig01.jpg"> 图1:阶段尖基磷蛋白法的工作流程。蛋白质从野生型和 snrk2-dec 突变幼苗中提取和消化，无论是否含有曼尼托。每个复制品的消化肽都标有独特的 TMT6-plex 通道。磷肽被汇集，然后使用 IMAC 舞台提示进行浓缩。纯化磷肽由 Hp-RP 阶段尖端分离。每个分数都由质谱仪分析。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61489/61489fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61489/61489fig02.jpg"> 图2:IMAC阶段尖端和HP-RP分馏对野生型和 snrk2 突变幼苗的磷蛋白分析。（A） 每分馏识别磷肽的数目。（B） 基于阶段尖端的 Hp-RP 色谱的分离效率。相邻分数之间的重叠由相邻分数中识别的相同肽的百分比表示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61489/61489fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61489/61489fig03.jpg"> 图3:对气溶胶应激的磷蛋白变化进行量化。火山地块显示（A）野生型和（B）snrk2-dec突变幼苗中磷化位点的原木2折叠变化，以响应曼尼托治疗。黑圆代表由曼尼托引起的激酶磷化位点。红色圆圈表示 RAF 的磷酸化位点因曼尼托而向上调节。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61489/61489fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>缓冲区 A</td>
			<td colspan="2">200 mM 铵（NH4HCO2），pH 10.0。</td>
		</tr>
<tr>
<td>缓冲区 B</td>
			<td colspan="2">100% 交流。</td>
		</tr>
<tr>
<td>缓冲区 1</td>
			<td colspan="2">5% 缓冲 B， 95% 缓冲区 A.</td>
		</tr>
<tr>
<td>缓冲区 2</td>
			<td colspan="2">8% 缓冲 B， 92% 缓冲区 A.</td>
		</tr>
<tr>
<td>缓冲区 3</td>
			<td colspan="2">11% 缓冲 B， 89% 缓冲区 A.</td>
		</tr>
<tr>
<td>缓冲区 4</td>
			<td colspan="2">14% 缓冲 B， 86% 缓冲区 A.</td>
		</tr>
<tr>
<td>缓冲区 5</td>
			<td colspan="2">17% 缓冲 B， 83% 缓冲区 A.</td>
		</tr>
<tr>
<td>缓冲区 6</td>
			<td colspan="2">20% 缓冲 B， 80% 缓冲区 A.</td>
		</tr>
<tr>
<td>缓冲区 7</td>
			<td colspan="2">23% 缓冲 B， 77% 缓冲区 A.</td>
		</tr>
<tr>
<td>缓冲区 8</td>
			<td colspan="2">80% 缓冲 B， 20% 缓冲区 A.</td>
		</tr>
</tbody></table>表1:Hp-RP阶段尖端分馏的缓冲区。

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

Nanyang Technological University

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

科研

生物化学

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

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 ...

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-&#946; peptide variants with their respective intrinsic aggregation propensities.

Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-&#946; peptide.

蛋白质聚集对酵母细胞氧化应激的影响评价

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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 a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

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 ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

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 ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...