导流洞得到了塞尔学者计划,. 史密斯基金会 (C1404) 和3月的硬币基金会 (1-财政年度-15-344) 的支持。R01GM110174 和 nih R01AI118891 以及国防部授予 BC123187P1 的支持。刻录-T。得到了 NIH 培训补助金 T32GM008216 的支持。

1. mESCs 的文化和扩展 注意: 小鼠胚胎干细胞易于培养, 可以迅速扩展到生物化学实验所需的大量时间, 这得益于它们的快速循环。健康的 mESCs 每12小时两倍. 
<ol> <li> 将 mESCs 扩展到所需的糊板上的制介质 (如下所示) 在37和 #176 的组织培养箱中; C、5% CO 2 和 #62; 95% 湿度. 
	注: 3-5 的生物复制为 + 4 su 样品和4SU 控制应准备足够的板材。一个10厘米的板块 ( 即 5-1000万细胞) 对于每个生物复制来说都足够了. </li>
	<li> mESCs 的培养基由 Dulbecco 和 #39; s 修饰的鹰和 #39; s 培养基 (DMEM), 补充15% 胎牛血清 (FBS), 2 毫米 l-谷氨酰胺, 0.5x 青霉素/链霉素, 1x 非必需氨基酸, 100 和 #181; M β-基, 1000 U/毫升白血病抑制因子 (LIF), 3 和 #181; CHIR99021, 1, #181; PD0325901. </li>
	<li> 在传代 mESCs 之前, 准备所需的 10 cm 板数. 
	<ol> <li> 在水中添加4毫升的0.1% 明胶, 确保覆盖整个表面; 在室温下孵育10分钟; 去除明胶溶液. </li>
	</ol> </li> <li> 通道单元格在每个板上达到 ~ 1000万单元格, 通常在接种后 48 h. 
	注: 当 mESCs 生长在稠密的菌落中时, 不应允许板块达到 100% 70-100, 如观察到生长在单分子膜上的细胞系 ( 如 HEK293 或3T3 细胞)。制板应传代当〜50% 汇合, 并明确之前, 介质变成黄色。
	<ol> <li> 要分离单元格, 用高密度制培养基在1x 磷酸缓冲盐水 (PBS) 中添加2毫米的 EDTA, 然后在 DMEM 中加入0.05% 的胰蛋白酶, 并在5分钟 (37 和 #176; C, 5% CO 2 , 和 #62; 95%湿度). </li>
		<li> 从孵化箱和重细胞中取出板, 移数次上下剧烈的 DMEM. </li>
		<li> 使用例计数单元格并确保单元格形成一个单细胞悬浮液 ( 即 无簇), 然后在室温下将细胞混合物 500 x g 离心5分钟。在 1-2 毫升 DMEM 中去除上清和重细胞颗粒. </li>
		使用步骤1.3.1 中制备的明胶涂层板, <li> 在密度为1万的细胞/cm 2 制培养基 ( 即每 10 cm 板上的 ~ 80万细胞) 中接种细胞. </li>
	</ol> </li> </ol> 2。蛋白质与 #8211 的交联; 活细胞中的 rna 相互作用 注意: rna-蛋白质的激活介导的 ribonucleoside 模拟 4-thiouridine (4SU). 4SU 具有比内源更长的吸光度核苷酸和只能纳入 RNA;因此, 中间波长的 UVB 可用于选择性地将 RNA 与蛋白质交联, 25 , 26 。UVB 治疗 4 su 处理的细胞导致共价通道4含苏 RNA 和氨基酸, 并报告偏好 Tyr, 跨国激进党, 满足, 赖氨酸, 和胱氨酸 27 . 
如步骤1所述, <ol> <li> 将 mESCs 扩展到所需的10厘米板数. 
	注: 3-5 的生物复制为 + 4 su 样品和4SU 控制应准备足够的板材。一个10厘米的板块 ( 即 5-1000万细胞) 是足够的每一个生物复制. </li>
	<li> 在板材到达70-100 之前, 将4SU 直接添加到介质中, 最终浓度为500和 #181; m. 离开-4SU 控制菜未经处理. </li>
	<li> 摇板轻轻地将4SU 均匀地分布在整个介质中。将它们返回到相同的组织培养孵化器2小时 (37 和 #176; C, 5% CO 2 , 和 #62; 95% 湿度). </li>
	<li> 将板移至冰桶并丢弃介质. </li>
	<li> 用5毫升的 ice-cold PBS 轻轻冲洗盘子. 
	注意: 轻轻冲洗或细胞可能分离. </li>
	<li> 添加2毫升的 ice-cold PBS 和放置无盖板的交, 配备有 UVB 发光灯泡 (312 nm)。照射板的能量设置为 1 J/厘米 2 . 
	注意: 这是至关重要的塑料盖被删除或他们可能保护细胞的紫外线和防止交联. </li>
	<li> 使用单元升降机收集细胞, 并将其转移到锥形管中的 ice-cold PBS。离心机在 2000 x g 为 5 min, 4 和 #176; C. </li>
	<li> 用2毫米 EDTA 和0.2 毫米磺氟化物 (PMSF) 去除5毫升 ice-cold PBS 中的上清和重细胞颗粒. </li>
	<li> 使用例计数单元格。预期分别为10厘米和15厘米的 5-10 和 10-2000万细胞. </li>
	<li> 2000 x g 离心5分钟, 4 和 #176; C. 
	注: 颗粒状细胞可立即用于步骤3或它们可以在液氮中闪光冷冻, 并储存在-80 和 #176; C 无限期。这是一个潜在的停止点. </li>
</ol> 3。细胞核的分离 注意: 细胞核被分离以去除细胞质蛋白, 并增加核蛋白的覆盖率。这一步可以取代其他形式的细胞分馏研究限制性商业惯例在不同的蜂窝舱室. 
<ol> <li> 准备冰冷缓冲器 A (10 毫米三盐酸 pH 7.9, 4 和 #176; C, 1.5 毫米氯化镁 2 , 10 毫米氯化钾)。使用至少2.5 毫升每1000万细胞 (在步骤2.9 中计算), 以提取. 
	注: 缓冲器 a (如500毫升瓶) 的库存可以提前准备, 并储存在4和 #176; C 为6月。
	<ol> <li> 在使用前, 将 0.5 mm 糖 (PMSF) 和 0.2 mm 的 ice-cold 缓冲区 A 的所需数量 (每1000万个单元格) 添加到2.5 毫升. </li>
	</ol> </li> <li> 每1000万单元格使用 2 mL 缓冲来清洗单元格。在4和 #176 旋转 2500 x g, 5 分钟; C. </li>
	<li> 丢弃上清, 添加缓冲 a 补充0.2% 的非离子, 变性洗涤剂, 如辛基-苯氧基双-乙醇 (见 材料表 ), 500 和 #181; L 每1000万细胞。在4和 #176 旋转5分钟; C. </li>
	<li> 2500 x g 离心5分钟. </li>
	<li> 清除上清液; 颗粒包括完整的无细胞质的细胞核. 
	注: 核可立即用于4步或闪速冷冻在液氮和储存在-80 和 #176; C 无限期。这是一个潜在的停止点. </li>
</ol> 4。细胞核的裂解 注意: 交联的细胞核在质谱-相容的缓冲液中裂解释放蛋白质和蛋白质-RNA 复合物. 
<ol> <li> 添加裂解缓冲液 (9 米尿素、100毫米三盐酸 pH 值8.0、24和 #176; C)、200和 #181; L 每1000万单元格。几种使用1/8 和 #34 的剪切染色质; 探头50% 振幅设置为 5-十年代. </li>
	<li> 旋转 1.2万 x g 为5分钟, 并收集175和 #181; L 上清, 以避免颗粒. 
	注: 100 及 #181; 我将用于下一个 RBR ID 步骤和剩余的75和 #181; 裂解液的 l 可以储存在-80 和 #176; C 供以后使用和/或西部印迹分析. </li>
	<li> 通过布拉德福德化验或类似技术测量蛋白质浓度 28 。将不同样品中的蛋白质浓度稀释到1毫克/毫升或最低的样品浓度, 使用适当数量的裂解缓冲液. 
	注: 从 1000万 mESCs 的细胞核中, 预计100和 #160; 200 和 #181; 蛋白质 g. </li>
</ol> 5。胰蛋白酶消化 注意: 蛋白质被消化生成多肽 suitable 为自下而上质谱 (MS) 分析. 
<ol> <li> 在核裂解液 (5 毫米决赛) 中添加 "", 在 RT 中孵育1小时. </li>
	<li> 从新鲜的库存中添加碘 (14 mM 决赛); 在黑暗中孵育30分钟. </li>
	<li> 稀释5倍体积50毫米 NH 的裂解液 4 小贩 3 </li>
	<li> 添加胰蛋白酶 (和 #181; g 蛋白: 和 #181; g 胰蛋白酶 = 200: 1); 孵育在37和 #176; 一夜之间. </li>
</ol> 6。多肽的脱盐 <ol> <li> 为每个示例准备一个 custom-made 阶段提示. 
	<ol> <li> 6.1.1. 将固相萃取 C18 材料放入200和 #181 的底部; L 吸管尖. </li>
		<li> 添加50和 #181; 在 C18 盘的顶部 R3 反相树脂。在1.5 毫升离心管内, 将舞台尖端放在离心适配器内, 并将接头放在带适配器的地方. </li>
		<li> 添加100和 #181; L 100% 乙腈至提示洗涤。离心机在 1000 x g 为1分钟, 以去除溶剂. </li>
		<li> 平衡50和 #181; L 0.1% 乙酸酸 (TFA)。离心机在 1000 x g 为 1 min. </li>
	</ol> </li> <li> 将100% 甲酸添加到已消化的蛋白质样品中, 以降低 pH 值到 2-3. 
	注: 这应该需要〜5和 #181; L 每1毫升稀释的肽样品, 但 pH 值应测量, 并增加更多的甲酸, 如果需要. </li>
	<li> 将样本加载到 custom-made 阶段提示, 在 1000 x g 上离心1分钟, 直到所有的样本都经过提示. </li>
	<li> 50 和 #181 清洗绑定多肽; 0.1% TFA。离心机在 1000 x g 为 1 min. </li>
	<li> 洗肽通过加入50和 #181; L 洗脱缓冲液 (75% 乙腈, 0.025% TFA) 到阶段尖端和离心机在 1000 x g 为1分钟, 迫使洗脱缓冲器的小费. </li>
	<li> 使用 150 x g 和 1-3 帕斯卡的真空离心机为1小时烘干样品 
	注: 干燥多肽可储存在-80 和 #176; C 无限期。这是一个潜在的停止点. </li>
</ol> 7。交联 rna 的去除 注意: 用核酸处理多肽以去除交联 rna. 
<ol> <li> 重50和 #181 中的颗粒; L 2x 核酸缓冲器 (100 mm NH 4 小贩 3 , 4 mm 氯化镁 2 ). </li>
	<li> 使用 280 nm 的吸光度测定肽浓度, 假定1毫克/毫升的肽为 A 280 1. 
	注: 预期浓度为1毫克/毫升. </li>
	<li> 将所有样品调整到1毫克/毫升或最低浓度使用2x 核酸缓冲. </li>
	<li> 准备50和 #181 的主组合; l H 2 O + 1 和 #181; 高纯度核酸 (#8805; 250 U) (参考材料 表 ) 每样. </li>
	<li> 取50和 #181; 多肽样品 l, 添加50和 #181; H 2 O + 核酸主组合. </li>
	<li> 孵育37和 #176; C 为1小时进行质谱分析. 
	注: 多肽现在已经准备好进行质谱分析。他们可以立即分析或存储在-80 和 #176; C 无限期。这是一个潜在的停止点. </li>
</ol> 8。纳米液相色谱、质谱和原始数据处理 注意: 由于4的 su 交联改变了肽的质量, 它们的离子在 LC-ms/毫秒中不依赖于交肽的强度,因此, 这似乎是减少了交联。这种减少的程度和一致性反映了每个肽 24 的蛋白质-RNA 交联程度. 
<ol> <li> 制备 hplc 缓冲器, 如下所示: 0.1% 甲酸在 hplc 级水中 (缓冲器 a); 0.1% 甲酸在 hplc 级乙腈 (缓冲 B). </li>
	<li> 如果有可用的纳米高效液相色谱, 请将纳米柱与以下规格连接: 内径75和 #181; m; 用 C18-AQ 颗粒填充; 长度 20-25 厘米. 
	注: 如果高效液相色谱可在较高流速下运行, 则使用适当的列大小表示流速。高流量色谱法降低灵敏度. </li>
	<li> 程序的 HPLC 法如下: 流速 250-300 nL/分钟; 梯度从2到28% 缓冲 b 在 120 min, 从28到 80% b 为下 5 min 和等 80% b 为 10 min. 
	注: 如果 HPLC 没有自动列平衡之前的样品加载, 启动程序与10分钟在 0% B 之前加载样本. </li>
	<li> 程序的 MS 采集方法执行数据相关的获取 (多哈回合) 作为标准的猎枪蛋白质组学实验。仪器的工作周期应该交替使用最强烈信号的串联 ms 扫描 (或 ms) 进行全 ms 扫描。使用蛋白质组学 MS 运行的最佳设置. 
	注意: 要取得自信的结果, 请使用能够在高分辨率模式下执行至少全 MS 扫描的仪器 ( 例如 orbitrap、飞行时间)。要精确量化, 请确保全 MS 扫描之间的间隔不超过3秒. 较长的工作周期可能会阻止提取离子谱的精确定义. </li>
	<li> 加载 1-2 和 #181; 将样品加到 hplc 柱上, 并按程序运行 hplc-质谱/ms 法。对于每个生物复制执行至少两个独立的运行, 并视为技术复制. </li>
	<li> 在 ms 之后, 将原始文件导入到蛋白质组软件包中, 通过萃取离子色谱识别 ms 光谱和肽的定量。我们推荐可以执行多个 MS 运行的色谱对齐的软件包 (请参见 材料表 ) 29 , 30 。
	<li> 如果该选项在软件套件中可用, 请启用和 #34; 在开始数据处理之前, 在运行和 #34 之间进行匹配. </li>
	<li> 分析完成后, 将多肽列表连同量化值一起导出. </li>
</ol>

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W., 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=SONAR+Discovers+RNA-Binding+Proteins+from+Analysis+of+Large-Scale+Protein-Protein+Interactomes.">SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.</a> Mol Cell. 64 (2), 282-293 (2016).</li><li>Bonasio, 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=Interactions+with+RNA+direct+the+Polycomb+group+protein+SCML2+to+chromatin+where+it+represses+target+genes.">Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes.</a> Elife. 3, e02637 (2014).</li><li>Kaneko, S., 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=Interactions+between+JARID2+and+noncoding+RNAs+regulate+PRC2+recruitment+to+chromatin.">Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.</a> Mol Cell. 53 (2), 290-300 (2014).</li><li>Kaneko, S., 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=Phosphorylation+of+the+PRC2+component+Ezh2+is+cell+cycle-regulated+and+up-regulates+its+binding+to+ncRNA.">Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.</a> Genes Dev. 24 (23), 2615-2620 (2010).</li><li>Saldana-Meyer, 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=CTCF+regulates+the+human+p53+gene+through+direct+interaction+with+its+natural+antisense+transcript,+Wrap53.">CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.</a> Genes Dev. 28 (7), 723-734 (2014).</li><li>Castello, 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=Comprehensive+Identification+of+RNA-Binding+Domains+in+Human+Cells.">Comprehensive Identification of RNA-Binding Domains in Human Cells.</a> Mol Cell. 63 (4), 696-710 (2016).</li><li>He, 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=High-Resolution+Mapping+of+RNA-Binding+Regions+in+the+Nuclear+Proteome+of+Embryonic+Stem+Cells.">High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells.</a> Mol Cell. 64 (2), 416-430 (2016).</li><li>Favre, 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=4-Thiouridine+photosensitized+RNA-protein+crosslinking+in+mammalian+cells.">4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells.</a> Biochem Biophys Res Commun. 141 (2), 847-854 (1986).</li><li>Hafner, 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=Transcriptome-wide+identification+of+RNA-binding+protein+and+microRNA+target+sites+by+PAR-CLIP.">Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.</a> Cell. 141 (1), 129-141 (2010).</li><li>Favre, A., Saintome, C., Fourrey, J. L., Clivio, P., Laugaa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thionucleobases+as+intrinsic+photoaffinity+probes+of+nucleic+acid+structure+and+nucleic+acid-protein+interactions.">Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions.</a> J Photochem Photobiol B. 42 (2), 109-124 (1998).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Tyanova, S., Temu, T., Cox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MaxQuant+computational+platform+for+mass+spectrometry-based+shotgun+proteomics.">The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.</a> Nat Protoc. 11 (12), 2301-2319 (2016).</li><li>MacLean, 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=Skyline:+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments.">Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.</a> Bioinformatics. 26 (7), 966-968 (2010).</li><li>Kondo, Y., Oubridge, C., van Roon, A. M., Nagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+human+U1+snRNP,+a+small+nuclear+ribonucleoprotein+particle,+reveals+the+mechanism+of+5'+splice+site+recognition.">Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.</a> Elife. 4, (2015).</li><li>Hafner, 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=PAR-CliP--a+method+to+identify+transcriptome-wide+the+binding+sites+of+RNA+binding+proteins.">PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.</a> J Vis Exp. (41), (2010).</li></ol>

作者没有什么可透露的。

我们描述一个详细的实验协议, 以执行 RBR ID 在 mESCs, 并在适当的修改, 在任何细胞, 可以纳入4SU 到 RNA。其他的细胞类型可能需要优化的方法, 以确保足够的信噪比。此外, 虽然本文所述的议定书侧重于核限制性商业惯例的审查, 但 RBR ID 技术应易于适应不同的细胞隔间, 如胞或特定的器, 使用不同的分馏战略.可能需要优化的参数包括4SU 浓度和并网时间以及 UV 交联的能量。这些参数对于确保 RNA 蛋白通道的有效形成, 并产生一个满意的信噪比是非常重要的。我们已经表明, 312 nm UVB 光是更有效的交联4含素 rna 与蛋白质相比, 365 nm UVA 通常使用的 PAR 剪辑32。直接比较的 RBR-312 nm 和 365 nm, 证明了 312 nm UVB 导致识别一个更大的部分已知的限制性商业惯例24。
还有两种方法可用于执行蛋白质组范围的限制性惯例识别。其中一个叫做 RBDmap, 它与 RBR 的 ID 相似, 因为它利用了 UV 交联, RBDmap 女士被用来识别限制性商业惯例, 并在 HeLa 细胞中映射它们的 RNA 结合活动23。这种方法依赖于对 RNA 结合位点附近的多肽的阳性识别, 依赖于两个连续的寡糖-dT 拉降, 这表明它的假阳性率可能低于 RBR ID。但是, "拉" 起伏只允许识别绑定到波利亚 + RNA 的限制性商业惯例, 并且需要大量的输入材料, 比 RBR ID 高出 10-100 倍。RBR-ID 可以执行, 只要5µg 的蛋白质每生物复制 (〜2µg 每技术复制), 可以收集从多达25万细胞。
第二种方法, 称为声纳, 依靠数据挖掘的大型蛋白质-蛋白质相互作用数据库, 以检测蛋白质, 经常 co-purify 与已知的限制性商业惯例, 其与这些限制性商业惯例的互动, 因此可能介导的 RNA18。这种巧妙的方法非常强大, 可以在不进行任何湿实验室实验的情况下识别新的限制性商业惯例, 只要有适当的数据库;然而, 它不能揭示互动网站, 因此是互补的, 但不是替代 RBR 的 ID。
总之, 我们的 RBR ID 技术可以识别新的限制性商业惯例和地图他们的 RBRs 与有限的输入样本量, 不需要任何生物化学纯化的蛋白质 RNA 交联的复合物。这项技术对被识别的限制性商业惯例的 rna 的类型没有任何假设, 因此可以映射绑定到 non-polyadenylated 的蛋白质的 rna 结合活动, 其中许多是编码的, 这表明 RBR ID 可能是一种有用的技术来研究编码 rna 的调控作用。

RBR ID 方法的目的是确定新的 rna 结合蛋白 (限制性商业惯例) 和映射他们的 rna 结合的区域 (RBRs) 与肽水平的决议促进 rna 结合的突变体的设计和研究生物和生物化学蛋白质-RNA 相互作用的作用。
RNA 在生物分子中是独一无二的, 因为它既能充当携带遗传信息的信使, 也可以折叠成复杂的三维结构, 其生物化学功能更类似于蛋白质的12。越来越多的证据表明, 编码 rna (rna) 发挥重要作用的各种基因调控和表观遗传途径3,4,5和, 通常, 这些管理功能是调解的演唱会与特定的 RNA 相互作用的蛋白质。特别重要的是, 一组相互作用的蛋白质最近被确定为强烈研究的长 ncRNA (lncRNA) Xist, 提供有价值的洞察如何这 lncRNA 介导的女性细胞 X 染色体失活6,7 ,8。值得注意的是, 这些 Xist 相互作用的蛋白质中有几个不包含任何规范的 rna 绑定域9, 因此它们的 rna 绑定活动不能根据它们的主序列单独被预测为在硅片中。考虑到数以千计的 lncRNAs 在任何给定的单元格10中表达, 有理由认为它们中的许多可能通过与尚未被发现的 RNA 结合蛋白 (限制性商业惯例) 的相互作用而行动。因此, 确定这些新的限制性商业惯例的实验战略将大大促进解剖 rna 生物功能的任务。
以往试图识别限制性商业惯例的经验依赖于波利亚 + RNA 选择耦合质谱 (MS)11,12,13,14,15。虽然这些实验在假定的限制性商业惯例中添加了许多蛋白质, 但通过设计他们只能检测到 polyadenylated 转录的蛋白质。然而, 大多数小 rna 和许多 lncRNAs 不是 polyadenylated 的。16,在这些实验中, 17及其相互作用的蛋白质可能会被遗漏。最近的一项研究应用机器学习蛋白质-蛋白质 interactome 数据库, 以识别蛋白质, co-purified 与多个已知的限制性商业惯例, 并表明这些经常性的惯例合作伙伴更有可能拥有 RNA 绑定活动18。然而, 这种方法依赖于挖掘现有的大型交互数据库, 并且只能识别可在变性条件下使用已知的限制性商业惯例 co-purified 的蛋白质, 从而排除不溶、膜嵌入和缺乏蛋白质.
将蛋白质鉴定为善意的限制性惯例通常不会自动产生关于蛋白质-RNA 相互作用的生物和/或生物化学功能的信息。为了解决这一点, 通常需要确定相互作用中所涉及的蛋白质域和氨基酸残基, 以便特定的突变体可以被设计成在每一种新的限制性条件下测试 RNA 结合的功能19,20. 我们的小组和其他人以前的努力使用重组蛋白片段和删除突变体来识别 RNA 结合区域 (RBRs)19,20,21,22;然而, 这种方法是 labor-intensive 和不相容的高通量分析。最近, 一项研究描述了使用质谱仪23以高通量方式绘制 RNA 结合活动的实验策略;然而, 这种方法依赖于双波利亚 + RNA 的选择, 因此与上面描述的限制性惯例识别方法具有相同的限制。
我们开发了一种技术, 称为 rna 结合区域识别 (RBR ID), 利用蛋白质 rna 光和定量质谱法来鉴定蛋白质和蛋白质区域与 RNA 在活细胞中的相互作用, 而不做假设在 RNA 多状态, 因而包括限制性商业惯例对波利亚-rna24。此外, 这种方法完全依赖于交联, 对蛋白质的溶解度或可达性没有要求, 因此适于在细胞膜内 (例如核包络) 或不太可溶性的隔间映射 RNA 结合活动 (例如核矩阵)。我们描述了对小鼠胚胎干细胞的细胞核进行 RBR 的实验步骤 (mESCs), 但稍加修改, 本协议应适用于多种细胞类型, 只要它们能有效地将4SU 从培养中.

<table>
	<tbody>
		<tr>
			<td>KnockOut DMEM</td>
			<td>Fisher Scientific</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, qualified, US origin</td>
			<td>Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution 200 mM</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100&#215;)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>ESGRO Leukemia Inhibitory Factor (LIF)</td>
			<td>EMD Millipore</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901</td>
			<td>Sigma</td>
			<td>PZ0162</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin solution,2% in water</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>4-thiouridine</td>
			<td>Sigma</td>
			<td>T4509</td>
			<td>50 mM stock in water</td>
		</tr>
		<tr>
			<td>Spectrolinker XL-1500</td>
			<td>Fisher Scientific</td>
			<td>11-992-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride</td>
			<td>Sigma</td>
			<td>78830</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CO-630</td>
			<td>Sigma</td>
			<td>542334</td>
			<td>Commercial form of octyl-phenoxy-polyethoxy-ethanol detergent</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>I6125</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>Empore solid phase extraction disk</td>
			<td>3M</td>
			<td>66883</td>
			<td></td>
		</tr>
		<tr>
			<td>OLIGO R3 Reversed - Phase Resin</td>
			<td>Fisher Scientific</td>
			<td>1133903</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma</td>
			<td>E8263</td>
			<td>High purity nuclease</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator Model 100</td>
			<td>Fisher Scientific</td>
			<td>discontinued</td>
			<td>updated with FB505110</td>
		</tr>
		<tr>
			<td>HPLC grade acetonitrile</td>
			<td>Fisher Chemical</td>
			<td>A955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Fisher Scientific</td>
			<td>W6 4</td>
			<td></td>
		</tr>
		<tr>
			<td>TFA</td>
			<td>Fisher Scientific</td>
			<td>A11650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Sigma</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma</td>
			<td>49199</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr>
			<td>ReproSil-Pur 18-AQ</td>
			<td>Dr. Maisch GmbH HPLC</td>
			<td>r13.aq.0003</td>
			<td>Packing material for HPLC column</td>
		</tr>
		<tr>
			<td>Capillary for nano columns (75 &#181;m)</td>
			<td>Molex</td>
			<td>1068150017</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant software</td>
			<td>Max Planck Institute for Biochemistry</td>
			<td></td>
			<td>Can perform chromatographic alignment of multiple MS runs</td>
		</tr>
	</tbody>
</table>

sample preparation for mass spectrometry-based identification of rna-binding regions

编码 rna 在几个核过程中起重要作用, 包括调节基因表达、染色质结构和 DNA 修复。在大多数情况下, 编码 rna 的作用是由蛋白质介导的, 其功能反过来受这些相互作用与编码 rna。与此相一致的是, 越来越多的涉及核功能的蛋白质被报告为结合 rna, 在少数情况下, 这些蛋白质的 rna 结合区域已经被映射, 通常是通过费力的 candidate-based 方法。
在这里, 我们报告一个详细的协议, 以执行高通量, 蛋白质组范围内的 rna 结合蛋白和它们的 rna 结合区域的无偏见的识别。该方法依赖于在细胞 rna 中加入 photoreactive 苷模拟, 其次是紫外介导的蛋白质-rna 交联, 以及质谱分析, 以揭示蛋白质组内的 rna 交联肽。虽然我们描述了小鼠胚胎干细胞的过程, 但该协议应该很容易适应于各种培养细胞。

图 1描述了 RBR ID 工作流。由于这种技术的交联效率相对较低, 因此在生物复制中考虑到观察效果 (P-值) 的损耗水平和一致性非常重要。图 2显示了 RBR ID 结果的火山图。重叠 RNA 识别主题 (RRM) 域的多肽显示出高度一致的损耗水平。RRM 域可用作 RBR 分析的正则控制。
RBR ID 可用于在活细胞中映射 RBRs。可以计算每种肽的 RBR-id 分数来估计 RNA 结合电位: RBR-id 分数 = 日志2(正常化 + 4 su 强度/规范化-4SU 强度) x (日志10(P-值)2。图 3显示了在 spliceosomal 亚基 U1-70k 的表面上投射的 RBR 图的分数。在 rna 接触附近的明亮的红色颜色, 从晶体结构确定, 表明正确辨认蛋白质-rna 互作用。
在确定新的限制性商业惯例及其 RBRs 时, 强烈建议采用一种独立的方法来确认它们的 RNA 结合活动。在我们以前的研究24中, 我们将 TET2 识别为一种新的限制性行为, 并将 RNA 绑定活动映射到 C 终端区域, 如图 4A所示, 通过在蛋白质的主要序列上绘制 RBR ID 评分。图 4B显示了对此新 RBR 的要求可以通过使用该小波变换序列执行 PAR 剪辑26来验证, 并将该信号与缺少预测 RBR 的突变体进行比较。此验证实验的附加控制和更详细的说明可在他的et al.201624。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56004/56004fig1.jpg"> 
图 1: RBR-ID 概述.鼠标干细胞处理4SU 或不 (1-2) 和辐照 312 nm 紫外线 (3)。细胞核分离 (4), 提取物与蛋白酶和核酸, 产生交联和 uncrosslinked 肽 (5)。共价 RNA 加在交联的站点改变了肽质量, 导致相应地减退 uncrosslinked 肽的质量光谱 (6, 箭头) 的强度。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56004/56004fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56004/56004fig2.jpg"> 
图 2: mESCs 中的蛋白质组范围内的 RBR ID在x轴和学生的t-测试 y轴上用于±4SU 处理 (312 nm) 上的多肽强度的日志折叠显示的火山图。多肽重叠标注的 RRM 域是蓝色的。HNRNPC 的 RNA 结合肽以红色突出显示。以前在他et al. 201624中发布。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56004/56004fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56004/56004fig3.jpg"> 
图 3: RBR-ID 映射 protein–RNA 交互的站点.放大晶体结构的 U1 snRNP (PDB id: 4PJO31) 显示蛋白质表面 color-coded 根据他们的 RBR-ID 比分和相互作用的 rna 为 U1-70K。以前在他et al. 201624中发布。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56004/56004fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56004/56004fig4.jpg"> 
图 4: 验证 TET2 的 RBR.(A) TET2 的主要序列和已知域;沿主序 (中) 绘制的平滑残留层 RBR-ID 分数;和方案的表位标记催化域片段 (CD) 和 RBR 删除 (CDΔRBR) 结构用于验证 (底部)。(B) 在 HEK293 单元格中瞬时表达的 TET2 CD 和ΔCDΔRBR的 PAR 剪辑。显影为32P 标记的 RNA (顶端) 和控制西部印迹 (底部)。以前在他et al. 201624中发布。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56004/56004fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions at JoVE.com

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

我们描述了一个协议, 以确定 rna 结合蛋白和地图他们的 rna 结合区域的活细胞使用紫外介导的光和质谱。

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

sample-preparation-for-mass-spectrometry-based-identification-rna

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.0K 次观看.  University of Pennsylvania Perelman School of Medicine. 我们描述了一个协议, 以确定 rna 结合蛋白和地图他们的 rna 结合区域的活细胞使用紫外介导的光和质谱。

视频: Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

科研

生物化学

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 ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

用奇米蛋白构建功能蛋白区的鉴定

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

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 ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

使用自动工作站进行质谱测量的等离子体样品制备

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

蛋白质组学设施提交和后续数据分析的TMT样品制备

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

冷冻电镜高温样品网格的制备

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...