作者感谢内森. 约翰逊在准备这些数字方面的协助。这项研究得到了由美国能源部生物和环境研究局 (基因组科学计划) 资助的泛组学计划的支持, 微生物 (薄荷) 实验室指导研究开发倡议在太平洋西北国家实验室, 以及国家卫生研究院国家环境卫生科学研究所 (R01 ES022190) 和 NIH (P42 ES027704)。KEBJ 感谢 R21 HD084788 为开发和验证新的多涨价可能提取技术提供财政支持。这项工作是在格雷斯威利环境分子科学实验室 (EMSL), 在太平洋西北国家实验室 (PNNL) 的能源部国家科学用户设施进行的。PNNL 是由巴特利在合同 DE-AC06-76RL01830 下为能源部管理的多方案国家实验室。

注: 非常潮湿的土壤可以在萃取前冻干, 而不损害萃取效果。湿土壤也可以使用, 但应考虑当添加试剂在特定比率。
注: 建议使用20克的干土重量每萃取, 这必须在两个50毫升管 (最大10克土壤每50毫升管)。抽取可以根据可用样品向上或向下缩放。
注: 干燥土壤样品可以通过3毫米筛筛, 以融汇和去除小的根和岩石。不要筛过潮湿的土壤样品, 因为样品会被卡在屏幕上。
1. 土壤细胞裂解和提取代谢物、蛋白质和脂类 (定时 ~ 1 d)
<ol><li>每20克土壤样品, 重10克入二个单独50毫升管子是甲醇或氯仿兼容。要非常小心的管选择, 因为氯仿将浸出大多数塑料, 污染的样品。尽可能使用玻璃或用聚丙烯或聚四氟乙烯 (PTFE) 制成的塑料管。 注意: 在初始称重和均匀化步骤中, 保持土壤在冰上, 尽可能的冷却。</li>
	<li>将10毫升氯仿冲洗过的不锈钢和石榴石珠加入每管。在冰上, 添加4毫升的冷超纯水 (见材料表用于净化系统) 到每个管 (一个样本分裂两个管) 和转移的样品在一个冰桶到通风罩。</li>
	<li>使用25毫升玻璃血清学吸管, 快速添加20毫升冰冷2:1 氯仿: 甲醇 (v/v)。 注意: 氯仿: 甲醇有严重的潜在的健康影响: 皮肤刺激, 可能的化学烧伤和对呼吸系统的刺激。它可能会影响肾脏, 肝脏和心脏。佩戴合适的防护眼镜、衣物和手套, 并始终在通风罩中工作。</li>
	<li>拧紧盖子和漩涡入解答。2:1 choloroform: 甲醇有助于分解原核生物细胞壁, 也钝化酶活性。</li>
	<li>如果可能的话, 将管子连接到50毫升管涡流附件和水平涡10分钟, 在冰箱内4摄氏度。然后, 把样品放在一个-80 摄氏度的冰箱里, 15 分钟, 以便完全冷却下来。</li>
	<li>使用探测器 sonicator 在通风罩内, 油脂实验每个样品与6毫米 (1/4) 探针在振幅60% 三十年代每个在冰上。 注意: 建议在 sonicating 时使用声音减弱的外壳和耳保护。电源和高频电缆均存在高压。用主动探针避免接触样品容器的底部或两侧;它可能会开裂或熔化的塑料。</li>
	<li>把样品放在-80 摄氏度的冰箱里15分钟, 因为 sonicating 可以产生大量的热量。</li>
	<li>重复步骤 1.5-1.7。 注: 确保样品在溶解过程中保持冷。</li>
	<li>离心样品在 4000 x g 为5分钟, 4 °c。此时, 样品将被分离到上部代谢物层, 蛋白质夹层, 和低脂层 (和剩余的碎片颗粒)。请参见图 1。</li>
	<li>把样品放在冰桶里的冰上, 然后用10毫升的玻璃血清吸管把上面的代谢物层从两个50毫升的管子中移到一个大玻璃瓶中。 注意: 避免将任何蛋白质层吸入吸管;如果需要, 留下一小层代谢物。</li>
	<li>稍微倾斜50毫升管释放蛋白质夹层, 使其自由漂浮后, 低脂层。使用干净的不锈钢平头实验室铲, 仔细舀两个蛋白质夹层, 并把它们放在一起成一个新的50毫升管。</li>
	<li>使用25毫升玻璃血清学吸管, 将较低的脂质层移到一个大玻璃瓶中。 注意: 尽量避免有抱负的土壤颗粒;然而, 一些土壤碎片和粒子渴望随着油脂将被删除在以后的时间。</li>
	<li>将透气膜置于代谢物和脂质玻璃瓶的顶端, 干燥在真空浓缩器中直到干燥 (脂质〜 4-5 小时, 代谢物过夜)。</li>
	<li>对蛋白质样品管和剩余的碎片颗粒管, 加入20毫升冰冷甲醇到每个和漩涡。这是对碎片丸, 以漂洗氯仿之前, 试图溶解任何可能的剩余蛋白质出碎片之前, 折腾它。</li>
	<li>离心机的碎片颗粒和蛋白质夹层在 4000 x g 为5分钟, 4 摄氏度, 然后醒酒甲醇成一个危险的废物容器内的通风罩。</li>
	<li>冷冻的蛋白质和碎片颗粒在液氮和干燥的冻干机 (与收集器温度可以-105 °c 由于甲醇有一个非常低的冰点-98 °c) 2 小时。 注: 不要过度干燥的颗粒, 因为他们将更难溶解。</li>
	<li>添加10毫升蛋白质增溶缓冲液 (4% SDS, 100mM dithiothreitol) 在50mM 三缓冲器, pH 8.0; 请参阅辅助试剂设置) 到蛋白质夹层管和20毫升的每个碎片颗粒。 注意: SDS 导致急性毒性, 是易燃的。它是皮肤, 眼睛和呼吸道刺激性。戴上手套和安全眼镜。</li>
	<li>在通风罩中, 探针油脂实验20% 振幅的样品在三十年代将其带入溶液中。涡流为2分钟。</li>
	<li>将蛋白质夹层样品放入实验室管转子中30分钟, 在 300 rpm, 50 摄氏度, 溶解蛋白质。</li>
	<li>水平涡旋的碎片样本额外的10分钟, 以溶解任何剩余的完整细胞, 然后旋转与蛋白质夹层样本的其余20分钟。</li>
	<li>离心机所有的样品在 4500 x g 10 分钟, 室温 (RT), 并收集上清从每个管每一个样本成两个50毫升管。</li>
	<li>将10毫升的增溶缓冲液添加到蛋白质夹层颗粒中, 然后油脂实验和涡旋回到溶液中, 并与前离心机一样。</li>
	<li>将上清液与两个50毫升管均匀地结合在一起, 离心机在 8000 x g 的10分钟, 4 °c, 在一个固定的角度斗转子。 注: 最终离心是清除过量污染腐植酸物质的必要条件。</li>
	<li>醒酒的上清液成两个50毫升管, 使每一个有30毫升。然后, 使用10毫升玻璃血清吸管, 添加7.5 毫升的 TCA (乙酸酸) 每管。漩涡变成溶液。这使 20% TCA 在每30毫升样品 (相应地调整), 注意: TCA 是刻薄的, 有毒的, 可能导致皮肤灼伤。戴上手套和安全眼镜。</li>
	<li>把样品放在-20 摄氏度的冰箱里, 2 小时到一夜之间。 注意: 蛋白质通常会在1小时内沉淀, 但可以隔夜 (最多18小时)。 注: 由于可能的酸水解蛋白质, 不要让 TCA 萃取超过18小时。如果样品结冰, 在冰上解冻;不要让样品预热过去解冻。</li>
	<li>为了将沉淀的蛋白质颗粒, 离心样品在 4500 x g 为10分钟, 4 °c 并且醒酒上清入废物。</li>
	<li>加入10毫升100% 冰冷丙酮到每个蛋白质颗粒, 漩涡和结合像小球成一管。</li>
	<li>离心管包含组合颗粒 (使用平衡), 然后醒酒上清成废物。 注意: 丙酮可能引起呼吸道和皮肤和眼睛刺激, 是易燃液体和蒸气。戴安全眼镜手套和实验室大衣, 在通风罩工作。</li>
	<li>使用1.5mL 丙酮清洗颗粒两次, 最后转到2毫升管, 最终旋转 1万 x 克5分钟。</li>
	<li>醒酒上清入废料, 并允许颗粒干倒在一个纸擦在一个通风罩20分钟或在氮气流, 直到颗粒轻微开始开裂。</li>
	<li>添加 100-200 µL 的蛋白质增溶缓冲, 取决于颗粒的大小。 注: 尽量降低添加到样品中的增溶缓冲容积。后续的过滤辅助样品准备 (FASP) 只能使用多达 50 ul 的可溶性样品每列;任何其他必须被拆分成多个 FASP 列。</li>
	<li>油脂实验和涡旋颗粒进入溶液。请注意, 样品可能是粘性的, 由于腐植物质沉淀随着蛋白质, 这将被删除与随后的离心。</li>
	<li>摇动样品在孵化器/振动筛为30分钟在 300 rpm, 40 °c, 溶解蛋白质入溶液和进行蛋白质消化。</li>
	<li>把样品冷冻在液氮中, 贮存在-80 摄氏度的冷藏库中, 直到准备好蛋白质消化。</li>
</ol>2. 脂质准备 (定时 ~ 20 分钟)
<ol><li>一旦脂质样品干燥, 添加 200 ul 2:1 氯仿: 甲醇到瓶子, 涡流成溶液, 并转移到1.5 毫升聚丙烯管, 增加一个额外的 200 ul 的玻璃瓶, 漩涡和吸管的剩余脂质和转移到管。</li>
	<li>将样品中的碎屑离心出 1.2万 x 克, 5 分钟4摄氏度。</li>
	<li>将上清液转移到玻璃脂瓶中, 储存在-20 摄氏度, 直到 lc-ms/毫秒分析 (在补充方法中的 lc ms/毫秒的其他详细信息)。</li>
	<li>如果样品在制备后不能立即进行分析, 脂质需要贮存在-20 摄氏度的溶剂中, 以防止氧化和降解。</li>
</ol>3. 代谢物的制备和衍生 (定时 ~ 5 h)
<ol><li>在提取后的第二天, 从速度 Vac 中取出代谢物样本, 并在-20 °c 处干燥, 直到准备好衍生和分析 GC。如果没有在 GC 上运行代谢产物, 则使用该仪器的适当协议准备它们。</li>
	<li>在 derivatizing 代谢物进行分析之前, 立即将其从大玻璃瓶中加入 200 ul 的甲醇, 涡流, 加入1.5 毫升聚丙烯管。重复一次。</li>
	<li>离心管在 1.2万 x g 10 分钟, 4 摄氏度。将上清液转移到较小的玻璃瓶中, 在顶部添加透气膜, 并在速度 Vac 中完全干燥。</li>
	<li>Derivatize 的代谢产物通过添加20µL 的 methoxyamine 溶液的样品瓶和漩涡三十年代在 vortexer 以中等速度。 注意: 盐酸 Methoxyamine 引起严重烧伤, 眼睛严重受损, 可引起皮肤接触致敏。佩戴安全眼镜, 手套和实验室大衣, 并在通风罩工作。</li>
	<li>使用浴 sonicator, 以确保样品完全溶解。</li>
	<li>孵化的样品在一个孵化器与凝结预防盖子保持在37°c 1 小时30分钟, 1000 rpm 晃动。</li>
	<li>将瓶子反转一次, 将样品与瓶盖表面的凝结水滴混合。在 1000 x g 处旋转样品1分钟, RT。</li>
	<li>用 1% trimethylchlorosilane 的 n-甲基 n-(三甲基硅) trifluoroacetamide 添加80µL (使用注射器) 进行硅烷化。漩涡10s。 注意: MSTFA + 1% 差旅管理公司可引起皮肤腐蚀, 严重的眼部损伤, 以及特定靶器官的毒性, 是易燃液体和水蒸气。佩戴安全眼镜, 手套和实验室大衣, 并在通风罩工作。</li>
	<li>孵化的样品在孵化器与凝结预防盖子保持在37°c 30 分钟, 1000 rpm 晃动。</li>
	<li>将瓶子反转一次, 将样品与瓶盖表面的凝结水滴混合。</li>
	<li>将样品向下旋转5分钟, 在 2000 x g, RT。</li>
	<li>将反应溶液转化为适合于 GC-MS 分析的小瓶。</li>
</ol>4. 蛋白质消化 (定时 ~ 1 d)
<ol><li>离心样品在 1.5万 x g 5 分钟, RT, 颗粒的任何碎片。</li>
	<li>使用 FASP 套件 (参见材料表) 执行筛选器辅助样例准备 (FASP), 按照修改后的制造商说明31,32:<ol>
<li>将8米尿素缓冲器的 400 ul 添加到 FASP 柱 (500 ul 30K 截留分子量旋转过滤器) 中。</li>
		<li>一旦样品完成离心, 去除上清液 (丢弃颗粒), 并增加 50 ul 的样品到 400 ul 8 米尿素在专栏。 注: 每列最多可添加50个 ul 样本。如果有超过 50 ul 的样品, 使用多列, 并结合在消化后的肽。最好把这个音量保持在尽可能低的 (30 µL 是理想的), 以确保 FASP 柱将删除所有的 SDS, 这可能会极大地干扰质量规范分析。</li>
		<li>将该列放入所含的1.5 毫升管中, 并将该样品离心在 1.4万 x g 处, 用于30分钟, RT。</li>
		<li>去除流动入废物并且再增加 400 ul 8 M 尿素对专栏和离心机。</li>
		<li>重复上一步一次, 总共3尿素漂洗。 注: 确保样品在柱上接近干燥, 如果每次旋转后在过滤器上残留30µL, 则继续离心。</li>
		<li>添加 400 ul 50 毫米 NH4HCO3和离心机在 1.4万 x g 为20分钟, 丢弃废物, 并重复一次。</li>
		<li>把柱子转移到干净的1.5 毫升离心管上。</li>
		<li>将胰蛋白酶消化缓冲剂的 75 ul 添加到柱上, 并在37摄氏度内孵育3小时, 用冷凝预防盖子在 750 rpm 处孵化。</li>
		<li>将40µL 50 毫米 NH4HCO3添加到列中。</li>
		<li>离心样品在 1.4万 x g 15 分钟收集的多肽进入管, 同时保持高分子量污染物在柱顶部。</li>
		<li>添加 40 ul 50mM NH4HCO3到列和离心机。</li>
	</ol>
</li>
	<li>丢弃该柱, 将试管中的多肽干燥至30µL, 并测定二辛可宁酸蛋白检测试剂盒; 请参阅材料表) 肽。</li>
	<li>(可选) 如果怀疑 SDS 污染 (可见气泡)33, 则执行清理步骤。如果选择此选项, 则必须事后进行固相提取。有关此清理方法的详细信息可在辅助方法中使用。</li>
	<li>稀释样品进行 MS 分析或选择进行 HPLC 分馏 (下两步).
</li>
	<li>分级, 稀释样品到400µL 的体积与10毫米甲酸铵缓冲 (pH 10.0)。</li>
	<li>在 C18 列上解析 (参见材料表), 方法是使用具有移动相的高效液相色谱系统 (A) 10 毫米甲酸铵, ph 10.0 和 (B) 10 毫米甲酸铵, ph 值 10.0/乙腈 (10:90)。<ol>
<li>调整渐变从 100% a 到 95% a 在前10分钟, 95% a 到 65% a 在极小的10到 70, 65% a 到 30% a 在极小的70到 85, 维护在 30% a 在极小的85到95。</li>
		<li>重新平衡与 100% A 在95到105并且保持在 100% A 直到极小的120。</li>
		<li>收集分数每1.25 分钟 (96 个分数在整个梯度), 最后合并每行总共12个样本或每其他行24样本 (每个有 n = 8 或 n = 4 分数汇集)。</li>
	</ol>
</li>
	<li>在真空下干燥所有馏分, 将15µL 的超纯水添加到-20 摄氏度, 直到 LC-ms/毫秒分析。</li>
</ol>

<ol>
	<li>Hultman, J., 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=Multi-omics+of+permafrost,+active+layer+and+thermokarst+bog+soil+microbiomes.">Multi-omics of permafrost, active layer and thermokarst bog soil microbiomes.</a> Nature. 521, 208-212 (2015).</li><li>White, R. 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=Moleculo+long-read+sequencing+facilitates+assembly+and+genomic+binning+from+complex+soil+metagenomes.">Moleculo long-read sequencing facilitates assembly and genomic binning from complex soil metagenomes.</a> mSystems. 1, (2016).</li><li>White, R. A., Callister, S. J., Moore, R. J., Baker, E. S., Jansson, 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=The+past,+present+and+future+of+microbiome+analyses.">The past, present and future of microbiome analyses.</a> Nat Protoc. 11, 4-8 (2016).</li><li>Ritchie, M. D., Holzinger, E. R., Li, R., Pendergrass, S. A., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+integrating+data+to+uncover+genotype-phenotype+interactions.">Methods of integrating data to uncover genotype-phenotype interactions.</a> Nat Rev Genet. 16, 85-97 (2015).</li><li>Jansson, J. K., Baker, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-omic+future+for+microbiome+studies.">A multi-omic future for microbiome studies.</a> Nat Microbiol. 1, (2016).</li><li>Domon, B., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Options+and+considerations+when+selecting+a+quantitative+proteomics+strategy.">Options and considerations when selecting a quantitative proteomics strategy.</a> Nat Biotechnol. 28, 710-721 (2010).</li><li>Marx, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+proteomics.">Targeted proteomics.</a> Nat Methods. 10, 19-22 (2013).</li><li>Roberts, L. D., Souza, A. L., Gerszten, R. E., Clish, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+metabolomics.">Targeted metabolomics.</a> Curr Protoc Mol Biol. , (2012).</li><li>Folch, J., Lees, M., Sloane Stanley, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues.">A simple method for the isolation and purification of total lipides from animal tissues.</a> J Biol Chem. 226, 497-509 (1957).</li><li>Nakayasu, E. 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=MPLEx:+a+Robust+and+Universal+Protocol+for+Single-Sample+Integrative+Proteomic,+Metabolomic,+and+Lipidomic+Analyses.">MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses.</a> mSystems. 1, (2016).</li><li>Bligh, E. G., Dyer, W. J. <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+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can J Biochem Physiol. 37, 911-917 (1959).</li><li>Pomraning, K. 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=Multi-omics+analysis+reveals+regulators+of+the+response+to+nitrogen+limitation+in+Yarrowia+lipolytica.">Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica.</a> BMC Genomics. 17, 138 (2016).</li><li>Tisoncik-Go, J., 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=Integrated+Omics+Analysis+of+Pathogenic+Host+Responses+during+Pandemic+H1N1+Influenza+Virus+Infection:+The+Crucial+Role+of+Lipid+Metabolism.">Integrated Omics Analysis of Pathogenic Host Responses during Pandemic H1N1 Influenza Virus Infection: The Crucial Role of Lipid Metabolism.</a> Cell Host Microbe. 19, 254-266 (2016).</li><li>Kyle, J. E., 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=Uncovering+biologically+significant+lipid+isomers+with+liquid+chromatography,+ion+mobility+spectrometry+and+mass+spectrometry.">Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry.</a> Analyst. 141, 1649-1659 (2016).</li><li>Lovelace, E. 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=Silymarin+Suppresses+Cellular+inflammation+by+inducing+reparative+stress+signaling.">Silymarin Suppresses Cellular inflammation by inducing reparative stress signaling.</a> J Nat Prod. 78, 1990-2000 (1990).</li><li>Kim, Y. 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=Diel+metabolomics+analysis+of+a+hot+spring+chlorophototrophic+microbial+mat+leads+to+new+hypotheses+of+community+member+metabolisms.">Diel metabolomics analysis of a hot spring chlorophototrophic microbial mat leads to new hypotheses of community member metabolisms.</a> Front Microbiol. 6, 209 (2015).</li><li>Pomraning, K. 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=Comprehensive+Metabolomic,+Lipidomic+and+microscopic+profiling+of+Yarrowia+lipolytica+during+lipid+accumulation+identifies+targets+for+increased+lipogenesis.">Comprehensive Metabolomic, Lipidomic and microscopic profiling of Yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis.</a> PLoS One. 10, e0123188 (2015).</li><li>Huang, E. L., 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=The+fungus+gardens+of+leaf-cutter+ants+undergo+a+distinct+physiological+transition+during+biomass+degradation.">The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation.</a> Environ Microbiol Rep. 6, 389-395 (2014).</li><li>Deatherage Kaiser, B. L., 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+Multi-Omic+View+of+Host-Pathogen-Commensal+Interplay+in+Salmonella-Mediated+Intestinal+Infection.">A Multi-Omic View of Host-Pathogen-Commensal Interplay in Salmonella-Mediated Intestinal Infection.</a> PLoS One. 8, e67155 (2013).</li><li>Kim, Y. 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=Salmonella+modulates+metabolism+during+growth+under+conditions+that+induce+expression+of+virulence+genes.">Salmonella modulates metabolism during growth under conditions that induce expression of virulence genes.</a> Mol Biosyst. 9, 1522-1534 (2013).</li><li>Ansong, 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=A+multi-omic+systems+approach+to+elucidating+Yersinia+virulence+mechanisms.">A multi-omic systems approach to elucidating Yersinia virulence mechanisms.</a> Mol Biosyst. 9, 44-54 (2013).</li><li>Bordbar, 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=Model-driven+multi-omic+data+analysis+elucidates+metabolic+immunomodulators+of+macrophage+activation.">Model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation.</a> Mol Syst Biol. 8, 558 (2012).</li><li>Hu, Z. 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=Metabolomic+response+of+human+skin+tissue+to+low+dose+ionizing+radiation.">Metabolomic response of human skin tissue to low dose ionizing radiation.</a> Mol Biosyst. 8, 1979-1986 (2012).</li><li>Perera, 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=Dengue+virus+infection+perturbs+lipid+homeostasis+in+infected+mosquito+cells.">Dengue virus infection perturbs lipid homeostasis in infected mosquito cells.</a> PLoS Pathog. 8, e1002584 (2012).</li><li>Gao, X., 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+reversed-phase+capillary+ultra-performance+liquid+chromatography-mass+spectrometry+(UPLC-MS)+method+for+comprehensive+top-down/bottom-up+lipid+profiling.">A reversed-phase capillary ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for comprehensive top-down/bottom-up lipid profiling.</a> Anal Bioanal Chem. 402, 2923-2933 (2012).</li><li>Sorensen, C. 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=Perturbations+in+the+lipid+profile+of+individuals+with+newly+diagnosed+type+1+diabetes+mellitus:+Lipidomics+analysis+of+a+Diabetes+Antibody+Standardization+Program+sample+subset.">Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: Lipidomics analysis of a Diabetes Antibody Standardization Program sample subset.</a> Clin Biochem. 43, 948-956 (2010).</li><li>Diamond, D. L., 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=Temporal+proteome+and+lipidome+profiles+reveal+hepatitis+C+virus-associated+reprogramming+of+hepatocellular+metabolism+and+bioenergetics.">Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics.</a> PLoS Pathog. 6, e1000719 (2010).</li><li>Alquier, T., 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=Deletion+of+GPR40+impairs+glucose-induced+insulin+secretion+in+vivo+in+mice+without+affecting+intracellular+fuel+metabolism+in+islets.">Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets.</a> Diabetes. 58, 2607-2615 (2009).</li><li>Ding, J., 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=Application+of+the+accurate+mass+and+time+tag+approach+in+studies+of+the+human+blood+lipidome.">Application of the accurate mass and time tag approach in studies of the human blood lipidome.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 871, 243-252 (2008).</li><li>Rasmussen, A. L., 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=Systems+virology+identifies+a+mitochondrial+fatty+acid+oxidation+enzyme,+dodecenoyl+coenzyme+A+delta+isomerase,+required+for+hepatitis+C+virus+replication+and+likely+pathogenesis.">Systems virology identifies a mitochondrial fatty acid oxidation enzyme, dodecenoyl coenzyme A delta isomerase, required for hepatitis C virus replication and likely pathogenesis.</a> J Virol. 85, 11646-11654 (2011).</li><li>Manza, L. L., Stamer, S. L., Ham, A. J., Codreanu, S. G., Liebler, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+and+digestion+for+proteomic+analyses+using+spin+filters.">Sample preparation and digestion for proteomic analyses using spin filters.</a> Proteomics. 5, 1742-1745 (2005).</li><li>Wisniewski, J. R., Zougman, A., Nagaraj, N., 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=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nat Methods. 6, 359-362 (2009).</li><li>Zhou, J. Y., 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=Simple+sodium+dodecyl+sulfate-assisted+sample+preparation+method+for+LC-MS-based+proteomics+applications.">Simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomics applications.</a> Anal Chem. 84, 2862-2867 (2012).</li><li>Anderson, J. 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=Decreased+abundance+of+type+III+secretion+system-inducing+signals+in+Arabidopsis+mkp1+enhances+resistance+against+Pseudomonas+syringae.">Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae.</a> Proc Natl Acad Sci U S A. 111, 6846-6851 (2014).</li><li>Chourey, K., 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=Direct+cellular+lysis/protein+extraction+protocol+for+soil+metaproteomics.">Direct cellular lysis/protein extraction protocol for soil metaproteomics.</a> J Proteome Res. 9, 6615-6622 (2010).</li><li>Kim, S., Gupta, N., Pevzner, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+probabilities+and+generating+functions+of+tandem+mass+spectra:+a+strike+against+decoy+databases.">Spectral probabilities and generating functions of tandem mass spectra: a strike against decoy databases.</a> J Proteome Res. 7, 3354-3363 (2008).</li><li>Kim, S., Pevzner, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MS-GF++makes+progress+towards+a+universal+database+search+tool+for+proteomics.">MS-GF+ makes progress towards a universal database search tool for proteomics.</a> Nat Commun. 5, 5277 (2014).</li><li>Cole, J. K., 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=Phototrophic+biofilm+assembly+in+microbial-mat-derived+unicyanobacterial+consortia:+Model+systems+for+the+study+of+autotroph-heterotroph+interactions.">Phototrophic biofilm assembly in microbial-mat-derived unicyanobacterial consortia: Model systems for the study of autotroph-heterotroph interactions.</a> Front Microbiol. 5, (2014).</li><li>Isaacson, T., 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=Sample+extraction+techniques+for+enhanced+proteomic+analysis+of+plant+tissues.">Sample extraction techniques for enhanced proteomic analysis of plant tissues.</a> Nat Protoc. 1, 769-774 (2006).</li></ol>

作者声明他们没有竞争的财政利益。

重要的是要注意的是, 并非所有的实验室都有相同的可用设备, 所以某些方法, 例如裂解步骤, 可以适应。在这里, 我们使用涡流和 sonicating, 但使用一个大型的50毫升珠搅拌器将工作。如果冻干机的集热器温度可为-105 摄氏度, 则可以在氮气流下干燥样品。土壤类型也有很大变化, 可以包括沙子、淤泥、粘土、泥炭和沃土 (等等), 它们也可以根据 pH 值、盐度和有机质的丰富程度而变化。这些差异...

评价土壤微生物群落对了解碳循环和气候变化具有重要意义。然而, 最近的研究突出了一些困难, 例如在不同土壤类型的微生物群中缺乏有序的基因组, 以及检测到的许多蛋白质的未知功能。这些挑战的结果是由于土壤是迄今为止已知的最复杂的微生物群落1,2,3。多涨价可能分析结合了宏基因组、metatranscriptomic、metaproteomic、metabolomic 和 lipidomic 研究的结果, 最近在许多土壤研究中得到了实施, 以更好地了解目前的微生物, 而获取有关由于环境扰动而发生的分子变化的全面信息1,4,5。多涨价可能研究的一个挑战是, 以质谱 (MS) 为基础的 metaproteomic、metabolomic 和 lipidomic 测量通常要求每个涨价可能的特定提取过程为 MS兼容6, 7,8,9. 当只有有限数量的样本可用时, 这些精确的程序使得它们的实施极其困难或不可能实现。这些挑战促使我们研究一种同时代谢物、蛋白质和脂质提取 (MPLEx) 方法, 能够使用体积较小的样本量或质量, 提高准确度, 并为所有三种分析提供更快的样品准备。10. 迄今为止, 没有任何替代土壤提取程序可以实现所有这些目标。
为了实现对单个土壤样品的全局多涨价可能分析, 采用了基于氯仿、甲醇和水分离的有机溶剂萃取协议 10.此方法最初是为总脂提取而开发的9,11和最近被修正了为同时提取代谢物, 蛋白质和油脂从一个样品12,13 ,14,15,16,17,18,19,20,21,22, 23,24,25,26,27,28,29,30, 启用较少的样本数量和实验性变异性10。在 MPLEx 协议中, 氯仿不与水混溶, 这为将样品组分三相化学分离为不同组分提供了依据。因此, 顶水相包含亲水性代谢物, 其次是蛋白质盘, 然后是底部氯仿阶段的脂质层 (图 1)。当 MPLEx 适用于大多数土壤时, 微粒碎片堆积在取样管的底部, 并且在收集所有层后可以被丢弃。然而, 每个土壤类型可以不同, 在高度有机土壤例如泥煤, 土壤残骸停留在中间层数并且不下落到取样管的底部。MPLEx 提供了几个优势, 当隔离多个分子类型从相同的样本, 如 1) 较小的样本量可用于多涨价可能分析, 2) 多涨价可能提取从相同的样本减少总体实验变异性, 并3) 可以更快地为更高吞吐量研究10编写更多的样本。这些好处对提供更好的测量能力以评估土壤样品及其复杂的微生物群落是至关重要的。

<table>
	<tbody>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>650498</td>
			<td>Stored at -20&#176;C !Caution chloroform has acute potential health effects, skin irritation and possible chemical burns, irritation to the respiratory system, may affect the kidneys, liver, heart. Wear suitable protective glasses, clothing and gloves, work in a fume hood.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Stored at -20&#176;C !Caution Methanol may cause respiratory tract, skin and eye irritation, may damage the nerves, kidneys and liver. Wear suitable protective glasses, clothing and gloves, work in a fume hood.</td>
		</tr>
		<tr>
			<td>Purified water from Millipore</td>
			<td>Milli-Q</td>
			<td></td>
			<td>Water purification system.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L6026</td>
			<td>!Caution SDS causes acute toxicity and is flammable. It is a skin, eye and airway irritant. Wear gloves and safety glasses.</td>
		</tr>
		<tr>
			<td>Soil protein extraction kit</td>
			<td>MoBio, NoviPure Soil Protein Extraction Kit, Qiagen</td>
			<td>30000-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Trizma HCL</td>
			<td>Sigma-Aldrich</td>
			<td>T2694</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T0699</td>
			<td>!Caution TCA is caustic, toxic and may cause skin burns. Wear gloves and safety glasses.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>650501</td>
			<td>Stored at -20&#176;C !Caution Acetone may cause respiratory tract and skin and eye irritation. Flammable liquid and vapor. Wear safety glasses gloves and a lab coat, work in a fume hood.</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>208884</td>
			<td>!Caution Urea is an eye and skin irritant, use gloves and safety glasses</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Fluka</td>
			<td>09830</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V528A</td>
			<td>20&#181;g vials</td>
		</tr>
		<tr>
			<td>Bicinchoninic acid protein assay kit</td>
			<td>Pierce</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Formate</td>
			<td>Sigma-Aldrich</td>
			<td>09735</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34998</td>
			<td>!Caution Acetonitrile is a skin and eye irritant. Highly flammable. Wear gloves and safety glasses. Work in a fume hood.</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508</td>
			<td>!Caution TFA is extremely hazardous in case of skin contact, eye contact, ingestion and inhalation. May produce tissue damage particularly on mucous membranes of eyes, mouth and respiratory tract. Skin contact may produce burns. Wear gloves, lab coat, safety glasses and work in a fume hood.</td>
		</tr>
		<tr>
			<td>Methoxyamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>226904</td>
			<td>!Caution Methoxyamine hydrochloride causes severe burns and serious damage to eyes, may cause sensitization by skin contact. Wear safety glasses, gloves and lab coat, work in a fume hood.</td>
		</tr>
		<tr>
			<td>Pyridine</td>
			<td>Sigma-Aldrich</td>
			<td>270970</td>
			<td>!Caution Pyridine can cause skin and eye irritation, central nervous system depression. Vapor may cause flash fire. Wear safety glasses, gloves and lab coat, work in a fume hood.</td>
		</tr>
		<tr>
			<td>N-Methyl-N-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane</td>
			<td>Sigma-Aldrich</td>
			<td>69478</td>
			<td>!Caution MSTFA + 1% TMCS can cause skin corrosion, serious eye damage and specific target organ toxicity. Flammable liquid and vapor. Wear safety glasses, gloves and lab coat, work in a fume hood.</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q water purification system</td>
			<td>Millipore</td>
			<td></td>
			<td>model MPGP04001</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Vortex Genie 2</td>
		</tr>
		<tr>
			<td>Probe sonicator</td>
			<td>FisherBrand</td>
			<td></td>
			<td>model FB505</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>model 5810R</td>
		</tr>
		<tr>
			<td>50mL tube swinging bucket rotor</td>
			<td>Eppendorf</td>
			<td>A-4-44</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL fixed angle rotor</td>
			<td>Eppendorf</td>
			<td>FA-45-6-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>OHAUS</td>
			<td></td>
			<td>model V22PWE150IT</td>
		</tr>
		<tr>
			<td>Serological pipette controller</td>
			<td>Eppendorf</td>
			<td>12-654-100</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL, 25mL glass serological pipettes</td>
			<td>FisherBrand</td>
			<td>13-678-27F, 13-678-36D</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer with Thermotop</td>
			<td>Eppendorf</td>
			<td>5382000015, 5308000003</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9 &#8211; 2.0 mm blend stainless steel beads</td>
			<td>NextAdvance</td>
			<td>SSB14B</td>
			<td></td>
		</tr>
		<tr>
			<td>0.15 mm garnet beads</td>
			<td>MoBio</td>
			<td>13122-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir plate</td>
			<td>FisherBrand</td>
			<td>11-100-16SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bar</td>
			<td>FisherBrand</td>
			<td>14512130</td>
			<td></td>
		</tr>
		<tr>
			<td>pH paper strips, pH range 0&#8211;14</td>
			<td>FisherBrand</td>
			<td>M95903</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL, 50mL conical polypropylene centrifuge tube</td>
			<td>Genesee Scientific</td>
			<td>21-103 21-108</td>
			<td>chloroform compatible</td>
		</tr>
		<tr>
			<td>50mL vortex attachment</td>
			<td>MoBio</td>
			<td>13000-V1-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>FisherBrand</td>
			<td>02-591-44</td>
			<td></td>
		</tr>
		<tr>
			<td>27.25x70mm glass vials</td>
			<td>FisherBrand</td>
			<td>03-339-22K</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe Easier plate membranes</td>
			<td>Midwest Scientific</td>
			<td>BERM-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol wipes</td>
			<td>Diversified Biotech</td>
			<td>BPWP-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Heater shaker incubator</td>
			<td>Benchmark, Incu-Shaker Mini</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analog rotisserie tube rotator</td>
			<td>SoCal BioMed, LLC</td>
			<td>82422001</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter-Aided-Sample-Prep kit</td>
			<td>FASP; Expedeon</td>
			<td>44250</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Biotek, EPOCH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-20 Degree Celsius Freezer</td>
			<td>Fisher</td>
			<td>13986149</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 Degree Celsius Freezer</td>
			<td>Stirling Ultracold</td>
			<td>SU78OUE</td>
			<td></td>
		</tr>
		<tr>
			<td>Q-Exactive ion trap mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 7890A gas chromatograph coupled with a single quadrupole 5975C mass spectrometer</td>
			<td>Agilent Technologies, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ-Orbitrap Velo</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waters NanoEquityTM UPLC system</td>
			<td>Millford, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250mL media bottle</td>
			<td>FisherBrand</td>
			<td>1395-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters vial</td>
			<td>Waters</td>
			<td>186002805</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass MS sample vial and inserts</td>
			<td>MicroSolv</td>
			<td>9502S-WCV, 9502S-02ND</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass HPLC vial and snap caps</td>
			<td>MicroSolv</td>
			<td>9512C-0DCV, 9502C-10C-B</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC 96-well plate</td>
			<td>Agilent</td>
			<td>5042-6454</td>
			<td></td>
		</tr>
		<tr>
			<td>Large glass vial 27.25x70mm</td>
			<td>FisherBrand</td>
			<td>03-339-22K</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7934021</td>
			<td></td>
		</tr>
		<tr>
			<td>Polished stainless steel flat head spatula</td>
			<td>Spoonula; FisherBrand</td>
			<td>14-375-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Kim wipes</td>
			<td>Kimberly-Clark</td>
			<td>34721</td>
			<td></td>
		</tr>
		<tr>
			<td>XBridge C18, 250x4.6 mm, 5 &#956;M with 4.6x20 mm guard column</td>
			<td>Waters</td>
			<td>186003117, 186003064</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1100 series HPLC system</td>
			<td>Agilent Technologies</td>
			<td>G1380-90000</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7mL centrifuge tube</td>
			<td>Sorenson</td>
			<td>11700</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Glass Syringes, 5mL, 50&#181;L and 250&#181;L</td>
			<td>Hamilton</td>
			<td>81517, 80975, 81175</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipettes</td>
			<td>FisherBrand</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipette Bulbs</td>
			<td>Sigma-Aldrich</td>
			<td>Z111597</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath Sonicator</td>
			<td>Branson 1800 Ultrasonic Cleaner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Centrifuge</td>
			<td colspan="2">Labconco Centrivap Acid-Resistant Concentrator System</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroSpin Columns, C18 Silica</td>
			<td>The Nest Group</td>
			<td>SEM SS18V</td>
			<td></td>
		</tr>
	</tbody>
</table>

the mplex protocol for multi-omic analyses of soil samples

以质谱 (MS) 为基础的综合 metaproteomic、metabolomic 和 lipidomic (多涨价可能) 研究正在改变我们对环境和生物系统中微生物群落的理解和表征能力。这些测量甚至能够增强对复杂土壤微生物群落的分析, 这是迄今为止已知的最复杂的微生物系统。然而, 多涨价可能分析确实有样本准备挑战, 因为每个涨价可能研究通常需要分离提取, 从而大大放大所需的准备时间和样本量。为了解决这一局限性, 通过调整溶剂基方法, 建立了从同一土壤样品中同时提取代谢物、蛋白质和脂质 (MPLEx) 的 3-1 方法。这种 MPLEx 协议已被证明是简单和健壮的许多样本类型, 即使是用于有限数量的复杂土壤样品。MPLEx 方法还大大启用了快速的多涨价可能测量, 以更好地了解每个微生物群落的成员, 同时评估发生在生物和环境扰动上的变化。

当 MPLEx 协议被用来从堪萨斯当地草原土壤中提取分子 (软土土壤) 时, 三项分析提供了3376多肽、105脂和102极性代谢物 (所有独特的证明) 的结果。虽然 MPLEx 协议已建立为一般提取脂质和代谢物12,13,14,15,16,17,<sup class="xre...

Watch this Scientific Journal Video about The MPLEx Protocol for Multi-omic Analyses of Soil Samples at JoVE.com

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

提出了一种从单个土壤样品中同时提取代谢物、蛋白质和脂质的协议, 允许缩短样品准备时间, 并对数量有限的样品进行多涨价可能质谱分析。

土壤样品多涨价可能分析的 MPLEx 协议

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

the-mplex-protocol-for-multi-omic-analyses-of-soil-samples

Research

JoVE Journal

Chemistry

10.8K 次观看.  Pacific Northwest National Laboratory. 提出了一种从单个土壤样品中同时提取代谢物、蛋白质和脂质的协议, 允许缩短样品准备时间, 并对数量有限的样品进行多涨价可能质谱分析。

视频: The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

科研

化学

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

表面修饰碳纳米管通过土柱运输

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important structural element found in many complex natural products and pharmaceuticals. Owing to the continual discovery of new guanidine-containing natural products and designed small molecules, rapid and efficient guanidinylation methods are of keen interest to synthetic and medicinal organic chemists. Because the nucleophilicity and basicity of guanidines can affect subsequent chemical transformations, traditional, indirect guanidinylation is typically pursued. Indirect methods commonly employ multiple protection steps involving a latent amine precursor, such as an azide, phthalimide, or carbamate. By circumventing these circuitous methods and employing a direct guanidinylation reaction early in the synthetic sequence, it was possible to forge the linear terminal guanidine containing backbone of clavatadine A to realize a short and streamlined synthesis of this potent factor XIa inhibitor. In practice, guanidine hydrochloride is elaborated with a carefully constructed protecting array that is optimized to survive the synthetic steps to come. In the preparation of clavatadine A, direct guanidinylation of a commercially available diamine eliminated two unnecessary steps from its synthesis. Coupled with the wide variety of known guanidine protecting groups, direct guanidinylation evinces a succinct and efficient practicality inherent to methods that find a home in a synthetic chemist&#39;s toolbox.

Here, we present a protocol for direct, early stage guanidinylation that enables rapid total synthesis of aminoguanidine-containing small organic molecules. An advanced synthetic intermediate used in the synthesis of a blood coagulation factor XIa inhibitor was prepared using this protocol.

一个直接的，早期胍基协议的复杂含氨基胍，天然产物合成

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

协议的合成<em&gt;邻</em&gt; -trifluoromethoxylated苯胺类化合物

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

一种醇溶蛋白氰基丙烯酸酯纳米粒子为亲水涂料生产协议

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

功能性降冰片烯的催化聚合插入一种简单高效的协议

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

使用有机锂试剂的一个协议安全的锂化反应

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

用于固相合成含有2&#39;-<em&gt; 0</em通过圆二色性修饰和表征

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.