门泽尔和 l. Ruess 的财政支持由德意志 Forschungsgemeinschaft (汝 RU780/11-1) 感激地承认。r. Nehring 由汝 780/10-1 资助。最后, 我们非常感谢榛 Ruvimbo Maboreke 博士校对我们的手稿。

被描述的协议不属于动物道德的能力。然而, 当人们将所描述的协议与更高的动物相适应时, 请注意动物伦理委员会批准了《牲畜处理议定书》。
1. 动物的耕种
注意: 所有解释的实验步骤都基于已建立良好的协议26,27,28。Biotests 在实验室需要持续供应容易可培养有机体。在这里, 使用了跳虫物种Protaphorura fimata (Gisin、1952) 和Hetermurus 大足(坦普尔顿, 1835)。这两种动物都是简单的维持作为生产的实验室文化喂养贝克的酵母。
<ol><li>在塑料缩影与紧的贴合盖子 (直径 7 cm, 高度 4.5 cm), 增加活性炭的混合物, 巴黎石膏和蒸馏水提供高度潮湿的养殖基体 (图 1A)。<ol>
<li>当准备缩影混合足够的基板时,例如,在批处理中为10缩影。将巴黎 (225 克) 和1部干活性炭 (25 克) 的石膏9个部分混合在一个石膏锅中, 小心地添加大约10个蒸馏水 (250 毫升), 并允许在室温下不搅拌的情况下坐5分钟。</li>
		<li>用实验勺子以顺时针方向搅拌, 避免气泡, 直到达到厚糊状的一致性。立即倒入缩影的高度约1厘米。</li>
		<li>平滑的石膏, 轻轻敲击板凳和旋转。注意, 孔 (气泡的随机产品) 和沟 (积极添加无菌铲) 可能会鼓励肥沃的跳虫产卵。这项研究避免了孔和沟有利于具有相同的可再生条件。但是, 为了演示目的,图 1B显示了一些漏洞。</li>
		<li>允许在室温下干燥 1-2 天;孵育在60°c 可能减少那时间到 1-2 h。</li>
		<li>在使用前先滋润缩影, 再用吸管加入自来水, 直到基体稍潮湿。保持微观湿润, 定期添加蒸馏水, 因为跳虫有一个柔软的角质层和容易脱水。</li>
	</ol>
</li>
	<li>使用简单的吸入管将跳虫容易地转移到石膏底座上,即长的硅管, 长约25厘米, 带有一个小网格的吸管尖端, 以防止动物吸入管内。或者, 通过允许它们附着在小刷子上的刚毛来转移动物。</li>
	<li>转移 (参见步骤 1.2) 30 新鲜地孵化了跳虫入新的缩影并且提供粒状干面包师的酵母作为食物 (关于刀子小费) (图 1B);每周至少更新两次。计划三独立复制每个取样日;在这个研究天 0-7 和14。在黑暗中孵化15摄氏度。保持恒温是必不可少的, 因为脂肪酸是由动物新陈代谢改变, 以满足膜流动性的要求。</li>
	<li>饲料跳虫与贝克的酵母四周前开始的暴露实验, 以获得一个均匀的13c/12c 信号和模式的脂肪酸。使用吸入管或刷子去除所有的鸡蛋 (图 1C), 粪便颗粒, 并定期蜕皮动物可能饲料, 从而改变他们的血脂剖面。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57110/57110fig1.jpg"> 图 1: 跳虫的种植.(A) 填充有繁殖基质的缩影, 一种由巴黎石膏、活性炭和蒸馏水混合而成的干燥混合物。(B) 和 (C) Protaphorura fimata区域性的代表性标本;注意干燥面包师的小掘金作为食物来源并且作为孔在繁殖基体 (黑箭头) (B) 并且二个蛋 (白色箭头) (C)。<a href="/files/ftp_upload/57110/57110fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 标签饮食、收获和样品处理
<ol><li>标签<ol>
<li>四星期在建立跳虫文化之后, 安置30个人入新的缩影和孵化在15°c 在黑暗中。</li>
		<li>通过将13C 标记的棕榈酸与贝克的酵母混合在一个比 0.5: 1,例如, 5 g, 给他们喂食面包师酵母包含完全13C 标记的棕榈酸 (13C16:0, 99 atom%), 引入脉冲标签13C16:0 和10克干面包师的酵母。在每个缩影上放置一个刀尖。</li>
		<li>6小时后, 用完全未标记的面包酵母取代这个标签食品。</li>
	</ol>
</li>
	<li>
进一步耕种
	<ol>
<li>在实验过程中, 每三天更新一次酵母的饮食, 增加的数量比跳虫在那个时期内消耗的要高。最重要的是, 使用吸入管或刷子清除 Collembolan 鸡蛋, 粪便颗粒和蜕皮定期, 以确保动物专属喂养的提供的食物。</li>
	</ol>
</li>
	<li>收获<ol>
<li>样品缩影破坏性每日直到天7。然后在14天, 收集三独立复制在每个取样时间;不同的取样时间是可能的。</li>
		<li>准备10毫升玻璃管, 配备聚四氟乙烯涂层螺丝帽, 每个样品一个。事先清洗这些管子在玻璃器皿洗涤器和漂洗两次与去离子水之后。最后, 通过添加2毫升氯仿 (HPLC 级)、涡流大致和废弃溶剂, 去除疏水性污染物的痕迹。</li>
		<li>对于控制样本 (0 天), 采取 3 30 非暴露跳虫从前文化作为天0样品。</li>
		<li>对于暴露的样品 (1 天和以后), 采取每日样品在每个案件 3 30 暴露的跳虫从文化 intermediately 哺养与13C 标记棕榈酸和贝克酵母的混合物。</li>
		<li>用超天平记录跳虫的新鲜重量。使用排或刷子将动物转移到适当的刻度盘上。为确保在秤盘中称量时容易处理跳虫, 震凉动物之前 (-80 °c 为2小时)。或者, CO2流为10分钟可以安全地电击动物。</li>
	</ol>
</li>
	<li>直接称量后把动物从鳞片锅里仔细地放入10毫升玻璃试管中。用1毫升甲醇 (HPLC 级) 填充管, 并贮存在-20 摄氏度, 直到分析。 注: 从这一步骤开始, 避免与塑料设备的样品处理, 因为有机溶剂涉及;改用适合于溶剂和玻璃容器的分配器和吸管。</li>
</ol>3. 动物组织和 Methanolysis 的脂质提取
<ol><li>准备三玻璃试管 (配备聚四氟乙烯涂层螺钉帽), 每批仅含1毫升甲醇作为空白值。重要的是, 添加或转让本协议中使用的任何溶剂仅由玻璃吸管或氯仿/甲醇冲洗耐溶剂的分配器。</li>
	<li>在油脂提取过程开始时, 通过蒸发降低用于贮存 (或冲裁) 的甲醇;推荐一种配备真空泵和冷阱的紧凑型台式旋转真空集中器 (RVC)。将开管转入 RVC, 蒸发至干燥50摄氏度, 真空压力为 200 hPa, 20 分钟。</li>
	<li>加入5毫升的单相萃取溶剂 (氯仿/甲醇/0.05 米磷酸盐缓冲 1:2: 0.8, pH 7.4) 到每个样品 (包括空白) 和提取 Collembolan 脂在室温下通过摇晃过夜 (~ 200 rpm)。</li>
	<li>将溶剂转移到新的管中, 再用2.5 毫升的萃取溶剂, 通过晃动3小时来重新提取样品。之后, 结合两个步骤的提取物;建议使用玻璃巴斯德吸管。添加0.8 毫升氯仿和0.8 毫升蒸馏水, 然后混合和离心机在2000克在20°c 为5分钟。最后, 允许样品为水和氯仿相分离提供5分钟的支持。</li>
	<li>对于脂肪酸模式分析, 将跳虫的总细胞脂分为中性脂质、糖脂宁和磷脂组分。<ol>
<li>为每个样品, 准备一个硅酸专栏 (商业专栏与 0.5 g 硅酸, 网格大小100-200 µm, 参见材料目录) 通过增加1毫升氯仿 (预处理)。为了加快这一过程, 在真空块上安装柱, 通常用于固相萃取色谱。不要使用尼龙针;用不锈钢管。</li>
		<li>在用于预处理的氯仿通过柱转移后, 将每个样品的氯仿的完全较低的一步转化为单个的柱。为了简化这一过程, 上部水相可以提前删除。使用吸管的玻璃, 但是, 注意列不干燥。</li>
		<li>先后洗脱5毫升氯仿 (包括中性脂脂肪酸, NLFAs), 10 毫升丙酮 (糖脂 cell-不分析在这个项目) 和5毫升甲醇 (包括磷脂脂肪酸, PLFAs)。收集各个玻璃容器中的每一个分数。</li>
		<li>在萃取结束时, 在 RVC 中通过蒸发减少氯仿 (NLFAs) 和甲醇 (PLFAs)。将开管转移到 RVC, 蒸发至干燥, 90 分钟60摄氏度, 真空度为 24 hPa。</li>
	</ol>
</li>
	<li>开始从韦尔奇 (1991)29中添加1毫升的氢氧化钠-甲醇溶液 (45 克氢氧化钠, 150 毫升甲醇和150毫升蒸馏水) 后的 NLFA 和 PLFA 馏分的脂肪酸皂化, 并孵化100在水浴中°c 30 分钟。在冰冷的水中冷却样品2分钟, 然后把样品放回长凳上, 继续在室温下工作。</li>
	<li>将内部标准添加到每个示例中, 包括空白。选择在实验生物体中不常见的脂肪酸;还可以使用饱和脂肪酸, 通过裂解和选择具有中间链长度的分子来最小化损失。对于许多用途, 奇数编号的 nonadecanoic 酸 (19:0) 工作良好。因此, 在异辛烷中添加30µL 的0.74 毫米溶液。确切的数量是非常重要的-一定要事先检查你的吸管的精确度与天平。</li>
	<li>加入2毫升盐酸-甲醇 (混合325毫升 6.0 N 盐酸与275毫升甲醇), 孵化在80°c 为10分钟在水浴和冷却迅速在冰为2分钟。这一步是时间和温度敏感;使用 80, 1 °c 和 10 @ 1 分钟检查多少样品可以进入水浴一次, 以保持80°c。 注: 本程序的结果是脂肪酸甲酯 (FAMEs),即,脂肪酸分析仪稳定的汽化气相色谱。</li>
	<li>最后, 加入1.25 毫升的己烷/甲基叔丁基醚 (1:1) 和岩石轻轻地10分钟, 然后离心机在2000克为5分钟. 移除底相, 保持顶相由 FAMEs 组成;使用玻璃巴斯德吸管。添加3毫升的水氢氧化钠 (10.8 克的氢氧化钠溶解在900毫升蒸馏水), 为洗涤步骤。<ol>
<li>岩石和离心机。</li>
	</ol>
</li>
	<li>使用玻璃巴斯德吸管完成上含脂的整个阶段, 并转移到装有聚四氟乙烯隔膜的气相色谱样品瓶中。避免包括少量的水相, 因为这将导致 GC 测量的问题。封装瓶和贮存在-20 °c, 直到分析。</li>
</ol>4. 用气相色谱法定量测定脂肪酸
<ol><li>用气相色谱法对跳虫 (动物) 脂的 NFLA 和 PLFA 馏分中的 FAMEs 进行鉴别和定量。气相色谱仪 (GC) 装有火焰电离检测器 (FID) 是一个已建立和证实的设备30。</li>
	<li>对于 FAMEs 的识别, 请将样本中峰值的保留时间与名望标准的比较。这些是质量或定量标准混合物的关键 FAMEs 覆盖各种食品和有机体。</li>
	<li>使用著名的标准混合物, 包括代表 FAs 的调查生物组和实验性饮食。在跳虫中, 这些都是真核生物的脂肪酸特征,例如, 长链多不饱和脂肪酸, 如花生四烯酸 (20:4ω6)。当使用酵母作为饮食这些真菌标记, 如亚油酸 (18:2ω6)。一个好的选择是所谓的名声混合, 包括37种不同脂肪酸频繁的动物, 真菌和植物材料, 和细菌酸甲酯 (BAME) 混合 (见材料表)。</li>
	<li>要在 GC 上运行示例, 请使用该仪器的相应软件设置一个序列。有关说明, 请参阅制造商手册。该序列从外部标准混合物 (例如名望和 BAME 混合) 开始, 后跟示例。请注意, 有一个保留时间移位,也就是说,在运行示例时, 每次运行时, 从 GC 列中的脂肪酸的洗脱时间略有延迟!无论是在示例序列中运行的每 10th或使用保留时间锁定棕榈酸 (16:0) 的标准。</li>
	<li>适应与仪器相关的 GC 设置。以下程序建议为高性能 (HP) 毛细管柱 (25 m x 0.2 毫米身份证, 膜厚度0.33 µm. 将注入量设置为1µL 分流进样模式, 并以氢气为载体气体。使用温度程序开始在50°c (举行1分钟) 和增加25°c 最小-1到175°c 后跟3°c 最小-1到230°c (举行为5.7 分钟)。</li>
	<li>计算 nmol 脂肪酸每克新鲜 (干) 重的有机体使用的反应为每个名望应用已知的数额为各自脂肪酸使用以下惯例: <img alt="Equation 1" src="/files/ftp_upload/57110/57110eq1.jpg"> 名望: 样品的各自名望的高峰区域 兆瓦FM: 各自名望的分子重量在µg 或µmol C是: µg 内部标准的集中度 是: 内部标准的峰值区域 m 列: 在 g 中各自跳虫样品的新鲜 (干) 重量 1000: 转换因素从µmol 到 nmol c生物武器: nmol 中相应空白值的平均值中各自名望的集中度</li>
</ol>5. 13C 通过 Isotopologue 剖析分析
<ol><li>使用一个 GC 系统耦合到一个质量选择检测器 (MS) 提供一个电子电离 (EI) 来源的 isotopologue 测定。<ol>
<li>使用极性毛细管柱 (例如, DB 23, CP-88), 因为这进一步允许分离不饱和脂肪酸, 即使有相同数量的双键。GC 列的选择对结果至关重要, 因为它决定了分子离子在脂肪酸中的良好表现。</li>
		<li>对于 DB 23 列 (60 m x 0.25 毫米的 id, 膜厚度0.15 µm), 开始烤箱温度在130°c 和增加6.5 °c/分钟到170°c。跟随以增量3°c/分钟到203°c 并且举行1.9 分钟跟随以40°c/分钟的增量到230°c 和举行8.3 分钟. 设置传输线温度为280摄氏度。再次, 调整 GC 方法的仪器。</li>
		<li>使用包括已知量的 FAMEs 的量化标准, 用于为13C 公司的所有脂肪酸进行调查。将这些标准放在每个示例序列运行的开始和结束处。从这些标准中取出感兴趣的脂肪酸的保留时间。</li>
		<li>测量样品从实验总是从未标记的样品开始, 并且标记的探针尔后。应用适合于样本浓度的拆分比率,例如1:12.5. 如果可用于仪器, 在每个样品运行后应用一个反冲, 以清除从剩余的分析仪中的柱。</li>
		<li>将保留时间锁应用于 GC-MS 方法, 使 SIM 采集不受移位, 且有可重现的分析物保留时间。</li>
	</ol>
</li>
	<li>使用该仪器的选定离子监测 (SIM) 模式, 确定13C 在脂肪酸的分子离子中加入。在 SIM 模式下的操作可以检测出与完全扫描模式相关的灵敏度增加的特定的方法。<ol>
<li>首先运行一个初始扫描, 看看有什么存在, 然后运行 SIM 在适当的离子。通过选择包含各自脂肪酸的色谱峰值时间的 m-/z 扫描窗口 (SIM 组), 获取分子质量感兴趣的数据。通常, 每个分析和时间窗口监控两到四个离子。</li>
		<li>为了提高灵敏度, 调整质量扫描速率和停留时间 (花在每个肿块上的时间)。最好的质量数据是以尽可能低的速度获得的, 而 SIM 中的一般规则是对分析物峰值的8到12扫描。仪表设置的代理是每质量9毫秒的平均停留时间, 周期为6秒, 扫描时间为 175 ms 周期-1。</li>
		<li>检测各自脂肪酸及其所有 isotopologues (m+1、m+2等) 的分子离子 (m+)。有关示例, 请参见具有代表性的结果。</li>
		<li>记录每个离子片段 (isotopologue) 的丰度。注意到分子离子的丰度和 isotopologues 的数量相对较低, 定量的质量很大程度上取决于 MS 系统的性能。在开始一个大样本序列 (实验) 之前运行一首曲子, 并在必要时清洁离子源。 注意: 首先, 这些数据通过消耗的前体 (这里 16:0) 产生每个脂肪酸的总的13C 充实。</li>
		<li>使用标记脂肪酸的同位素组成与未标记的对应物的比例来分配营养碳通量。使用步骤6.1 中的 atom% 公式计算标记碳 (原子百分比, atom%) 在各自脂肪酸中的百分比。将13c 中的跳虫脂肪酸的百分比与标签 (1 天和更高版本) 和未标记动物 (0 天) 的比例进行比较, 作为13C 从饮食到消费者流动的相对指示。</li>
		<li>将13C 的位置分配到脂肪酸链中。根据 isotopologues 的分布, 将整个标记示踪脂肪酸 (这里 16:0) 的膳食路由从链伸长的脂质代谢中解开。虽然整个标记分子的同化增加了最远的 isotopologues 的丰度到父离子 (M+),例如, m+15, m+16, 与链伸长由使用13C 标记 C2片段 (13C 乙酰 CoA) isotopologues 接近分子离子变得更加频繁。</li>
	</ol>
</li>
</ol>6. 13C 富集的计算
<ol><li>根据 isotopologues 的分布情况, 使用以下关系计算标记碳 (atom%) 在各自脂肪酸中的总百分比: atom% = (比率13C isotopologue 合并) x (频率各自 isotopologue) 这是在 Kuppardt et之后计算的。31为:<img alt="Equation 2" src="/files/ftp_upload/57110/57110eq2.jpg"> 其中N是脂肪酸中的碳原子数, J是13C 同位素的数量, M + J 是各自 isotopologue 的丰度, T 所有 isotopologues 的总丰度。</li>
	<li>计算时, 用 SIM-MS 分析法检测到各自 FA 和所有 isotopologues (m+1、m+2等) 的分子离子 (m+) 的峰值面积值, 并将其设置为100% 相对丰度。按照三的规则, 计算每个检测到的 isotopologue 的部分很容易。</li>
	<li>从实验值中减去非标记的天0控制值 (自然13c 背景), 以获取只追溯到执行的外部13C 标记的最终数据。</li>
</ol>

<ol>
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Isotopologue 分析
对 FAs 中13C 分布的数量方面的详细分析需要尖端技术来分配食物网中的碳分割。目前的工作使用 isotopologue 分析来评估热带相互作用的共同 FA 生物标志物的13c/12c 比值。采用液相色谱法 (LC) 对氨基酸进行分析, 并应用于致病细菌的碳代谢研究17,23。直到最近, isotopologue 的分析进一步发展为一个工具, 以研究门泽尔的小土壤无脊椎动物的脂质的饮食路线. 24由于此方法不受13C 信号的 "结转" 的影响, 因此, 与 GC--c IRMS32相比, 对具有特定同位素的有机组织的评价是有利的。
利用该方法, 用 EI 和 SIM 模式测定了分子离子及其 isotopologues 中的质量-电荷 (m-/z) 值。与完全扫描相比, SIM 中的灵敏度可以增加约 100, 甚至超过了 GC-FID 量化范围33。此外, 通过在给定时间窗口中获得指定的一组离子, 尽管共洗脱峰的背景为30, 但对分析物进行可靠的测量。Thurnhofer 和维特34证明, SIM 既适用于在食品样品中的 FA 鉴别和定量, 也适合于少量的脂质。
当使用13C isotopologue 的 SIM 时, 至少需要考虑两种方法约束。首先, 分子离子的丰度相对较低, isotopologues 更少。有了一个肮脏的离子源, 这些就变得不那么丰富了, 因此 SIM 方法只能是半定量的33。其次, 在 FA 的一个或多个碳位置上增加的13c 的出现减少了相关碳原子的13c 的自然丰度。这些变化与标签是非线性的, 一种现象叫 "倾斜" 在自然丰盈, 可能导致低估在这些大量的富集20。必须使用基于名望标准的更正矩阵 (参见费尔南德斯et), 或者将自然控制、即FAs 从没有标签的实验有机体中包括在内, 来解释这种扭曲效应。后者是在目前的工作进行比较相同的 FA 在标签 (1 天和更晚) 和非标签跳虫 (0 天)。
膳食路由和13C 通量
虽然 isotopologue 分析在 FAs 中的13C 测定的方法学优势是明显的, 但在体内的活体代谢可能受到生物手段的阻碍。同位素和代谢稳态是很难实现的, 当,例如, 饮食使用或消费者的生理状态不知道, 阻碍获得绝对碳通量。然而, 通过比较在饮食和消费者中 FAs 的13C isotopologues 剖面, 可以获得足总同化和代谢途径的相对比例。这种方法允许跟踪标记法的特定命运, 基于其13C 标签, 追踪其膳食路由。MS-SIM 表明, 标记法主要是路由到两个跳虫物种的 NFLA 分数分析24。此外, 标记法储存在中性脂质中, 只有轻微或无修饰。检测到的仅是膳食13C16:0 到18:0 和饱和到 monoenoic 等价项的伸长率 (请参见图 4)。在1天的 C16 和 C18 fas 中, 离子 M+16的存在证实了这些 FAs 是被补充的全标记棕榈酸的后代。这一发现支撑了早期的研究, 这是基于消费和饮食的足总模式仅5,35 , 并建议标记 FAs 作为整个分子纳入消费组织 (审查, 见 Ruess 和张伯伦8)。
同时, PLFAs 的 isotopologue 13C 模式显示, 随着时间的推移, 13C20:46 的观察到的增加是由于完全标记的前体 FAs 的伸长/饱和, 以及与后者的合成, 后者也包括13C 标记为乙酰 CoA, 如在 C20 中存在 M+18离子跟踪, 也 C18 FAs (请参见图 4)。图 5总结了被观察跳虫物种 PLFAs 和 NLFAs 中补充的13C16:0 的膳食路由。有趣的是, 只有13C 棕榈酸的 PLFAs 是受更强的变化, 而 NLFAs 的新陈代谢变化仍然只限于一个伸长和两个饱和步骤。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57110/57110fig5.jpg"> 图 5: 饮食的命运13C 棕榈酸在消费者油脂.流程图显示了被调查的跳虫物种中合并的13C16:0 的拟议命运。箭象征不同层次的新陈代谢更替不同的厚度。<a href="/files/ftp_upload/57110/57110fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
综上所述, isotopologue 分析清楚地表明, FA 作为营养标记是一种稳健可靠的方法, 在生态的食物网。虽然实验设计不允许精确量化的13C 通量 (例如,作为 atom% 13C 的补充 fa) 的不同步骤, 在 FA 代谢的途径, isotopologue 分析是一个有用的工具,所有生态研究使用脂肪酸作为营养标记。未来的目标是通过使用封闭的微观系统和喂养消费者定义的脂肪酸的前体数量来获得定量的消费法碳预算。

在一个神秘的栖息地如土壤, 营养关系是难以解决, 并进一步限制小规模的动物。在过去的十年中, 生物化学生态学取得了进展, 特别是在使用脂肪酸作为生物标志物, 以确定田间条件下土壤动物的饲养策略1,2,3。这是基于一个事实, 即从资源中脂肪酸可以被纳入到消费组织作为整个分子, 一个过程称为膳食路由4。从真菌到线虫到跳虫 5, 已经报告了三多种营养水平的脂肪酸转移。最近, 捕食动物区系被认为是6,7和第一次关于脂肪酸的评论作为土壤食物网的营养标记被发布了8,9。
通过脂肪酸稳定同位素探针 (FA-SIP) 获得了更详细的营养相互作用信息。在饮食和消费者中脂肪酸的13c/12c 比值的确定可以归因于二进制链接和估计伴生的碳流动, 并且被使用了在地球、淡水和海洋食物网10,11 ,12,13。基本的假设是, 膳食路由脂肪酸不受酶过程的影响;因此, 它们的13C 信号,即 13c/12c 比脂肪酸, 在消费者中类似于饮食1。然而, 在水生系统中报告了食物链的13C 签名逐渐枯竭, 从而阻碍了在营养研究中广泛应用 FA SIP14,15,16。此外, 陆地食物网中大多数无脊椎动物的脂代谢知识仍然有限。
了解消费者的脂质代谢通路, 对于利用营养标记脂肪酸作为测定食品网络生态学中定量碳流量的手段是至关重要的。考虑到这一点, 13C-isotopologue 分析, 原则上可以应用于对任何生物系统的碳代谢的研究17, 这是一个有希望的方法。在引入13c 标记的碳基板之后, 在新陈代谢网络中的13C 的分布是可追踪的, 因为在消费者中产生的代谢产物显示了特定的 isotopologue 分布。这可以通过定量的核代谢共振光谱学18,19或质谱20,21来评估, 后者在生物样品中偏爱低生物量由于其较高的灵敏度。
虽然 isotopologue 分析已成功应用于氨基酸, 并提供了对细菌病原体的体内碳代谢的洞察力17,22,23, 它的实施在脂肪酸已经落后了。第一次详细分析了稳定同位素标签前体脂肪酸的命运, 其膳食路由或降解通过β氧化, 在土壤无脊椎动物的消费者, 最近进行了门泽尔et 等。24. 在这里, 给出了与13C 标记脂肪酸结合实验的方法学基础, 并对常见的土壤无脊椎动物 (跳虫) 中的关键子代进行了 isotopologue 分析。这些 microarthropods 是一个很好的模型组, 因为它们构成了土壤食物网的重要组成部分, 并对其营养标记脂肪酸8,25进行了良好的调查。
了解消费者的脂质代谢通路, 对于利用营养标记脂肪酸作为测定食品网络生态学中定量碳流量的手段是至关重要的。本协议给出了实验室饲养实验的设计和建立, 以及从跳虫中提取和甲基化显性脂质分数 (中性脂质、磷脂) 的生物化学程序。通过质谱分析, 说明了脂肪酸的 isotopologue 组成, 并描述了相关的公式和计算。此过程的结果是: (i) isotopologues 超过父离子质量的比率 (即,脂肪酸分子离子 M+) 由一个或多个质量单位 (m+1, m+2, M+3,等) 和 (二) 总体13c. 从13C 标记的前驱体中提炼出的脂肪酸。虽然使用跳虫, 这种方法一般可以适用于任何其他捕食者-猎物互动的前提下, 这些是可培养在足够数量的控制条件下, 以确保成功的标签摄取和随后验证。

<table>
	<tbody>
		<tr>
			<td>neoLab-Round jars</td>
			<td>neoLab</td>
			<td>2-1506</td>
			<td>69 x 40 mm, 10 pacs/pack</td>
		</tr>
		<tr>
			<td>Charcoal activated</td>
			<td>Carl Roth</td>
			<td>X865.1</td>
			<td>p.a., powder, CAS No. 7440-44-0</td>
		</tr>
		<tr>
			<td>Alabaster Dental</td>
			<td>R&#214;HRICH-GIPSE</td>
			<td>---</td>
			<td>http://www.roehrich-gipse.de/dentalgipse.php</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Carl Roth</td>
			<td>7331.1</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>P717.1</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>Hexan</td>
			<td>Carl Roth</td>
			<td>7339.1</td>
			<td>HPLC &#8805; 98 %</td>
		</tr>
		<tr>
			<td>tert-Butyl methyl ether (MTBE)</td>
			<td>Carl Roth</td>
			<td>T175.1</td>
			<td>HPLC &#8805; 99,5 %</td>
		</tr>
		<tr>
			<td>Aceton</td>
			<td>Carl Roth</td>
			<td>7328.2</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Carl Roth</td>
			<td>6771.1</td>
			<td>p.a. &#8805;99 %, in pellets</td>
		</tr>
		<tr>
			<td>di-Natriumhydrogenphosphat</td>
			<td>Carl Roth</td>
			<td>P030.1</td>
			<td>p.a. &#8805;99 % , water free</td>
		</tr>
		<tr>
			<td>Na-dihydrogenphosphat Dihydrat</td>
			<td>Carl Roth</td>
			<td>T879.1</td>
			<td>p.a. &#8805;99 %</td>
		</tr>
		<tr>
			<td>Hypochloric acid (6 N)</td>
			<td>VWR International</td>
			<td>26,115,000</td>
			<td>AVS TITRINORM vol. solution</td>
		</tr>
		<tr>
			<td>Bond Elut (Columns)</td>
			<td>Agilent Tech.</td>
			<td>14102037</td>
			<td>HF Bond Elut-SI, 500 mg, 3 mL, 50/PK</td>
		</tr>
		<tr>
			<td>Pr&#228;paratengl&#228;ser Duran</td>
			<td>Glasger&#228;tebau Ochs</td>
			<td>135215</td>
			<td>&#216; 16 x 100 mm, plus screw cap with handy knurl and integrated PTFE/silicone gasket</td>
		</tr>
		<tr>
			<td>Supelco 37 Component FAME Mix</td>
			<td>Sigma-Aldrich</td>
			<td>47885-U Supelco</td>
			<td>10&#160;mg/mL in methylene chloride, analytical standard</td>
		</tr>
		<tr>
			<td>FlowMesh</td>
			<td>Carl Roth</td>
			<td>2796.1</td>
			<td>Polypropylene mesh, approximately 0.3 mm thick, with 1 mm strand spacing</td>
		</tr>
		<tr>
			<td>Bacterial Acid Methyl Ester (BAME) Mix</td>
			<td>Sigma-Aldrich</td>
			<td>47080-U Supelco</td>
			<td>10&#160;mg/mL in methyl caproate, analytical standard</td>
		</tr>
		<tr>
			<td>Methyl nonadecanoate</td>
			<td>Sigma-Aldrich</td>
			<td>74208</td>
			<td>analytical standard &#8805; 98.0 %</td>
		</tr>
		<tr>
			<td>Hexadecanoic acid-1-13C (Palmitic)</td>
			<td>Larodan Fine Chemicals</td>
			<td>78-1600</td>
			<td>GC &#8805; 98.0 % (13C: 99.0 %)</td>
		</tr>
		<tr>
			<td>RVC 2-25 CDplus</td>
			<td>Martin Christ Gefrier-trocknungsanlagen</td>
			<td></td>
			<td>Compact benchtop midi concentrator</td>
		</tr>
		<tr>
			<td>Alpha 2-4 LDplus</td>
			<td>Martin Christ Gefrier-trocknungsanlagen</td>
			<td></td>
			<td>Drying manifold</td>
		</tr>
		<tr>
			<td>MZ 2C NT</td>
			<td>Vacuubrand GMBH</td>
			<td></td>
			<td>Vacuum pump</td>
		</tr>
		<tr>
			<td>Roto-Shake Genie</td>
			<td>Scientific Industries</td>
			<td></td>
			<td>Combined rocking and rotating device</td>
		</tr>
		<tr>
			<td>XP64 Micro Comparator</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>Super high precision balance</td>
		</tr>
		<tr>
			<td>GC-System 7890A</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Gas chromatograph</td>
		</tr>
		<tr>
			<td>7000 GC/MS Triple Quad</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Triple Quad mass spectrometer</td>
		</tr>
		<tr>
			<td>7683B Series Injector</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Sample injector</td>
		</tr>
		<tr>
			<td>Heraeus Multifuge 3SR+</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Centrifuge with 10 ml tube rotor</td>
		</tr>
	</tbody>
</table>

fatty acid 13c isotopologue profiling provides insight into trophic carbon transfer and lipid metabolism of invertebrate consumers

脂肪酸 (FAs) 是食品网络生态学中有用的生物标志物, 因为它们通常被同化为一个完整的分子, 并转移到使用小的或没有修改的消费者组织中, 从而允许不同营养水平之间的膳食路由。然而, 由于对土壤动物脂质代谢的知识有限, 足总营养标记方法仍受到阻碍。本研究使用全标签棕榈酸 (13C16:0, 99 atom%) 作为示踪剂在脂肪酸代谢通路的两个广泛的土壤跳虫, Protaphorura fimata和Heteromurus 大足。为了研究这种前驱体的命运和代谢修饰, 提出了一种利用单离子监测的质谱法进行 isotopologue 分析的方法。此外, 还介绍了上游实验室饲养实验, 以及显性脂质组分 (中性脂质、磷脂) 的提取和甲基化, 以及相关的配方和计算。Isotopologue 分析不仅会产生从13c 标记为前体的脂肪酸中的总的13c 富集, 而且还产生超过母体离子质量的 isotopologues 的模式 (即, FA 分子离子m+) 每个标记为 FA 的一个或多个质量单位 (m+1, m+2, m+3,等等)。这一知识允许对完全消耗的 FA 的膳食路径与从头生物合成的比值进行结论。isotopologue 分析被认为是评价土壤动物脂肪酸代谢的有用工具, 以解开营养的相互作用。

跳虫的鲜重和脂质含量 在所述实验过程中, NLFAs 和 PLFAs 的含量在一段时间内没有显著变化, 而试样的新鲜重量略有增加, 但不显著24。两个参数都表明跳虫标本的身体素质良好。注意调查跳虫的新鲜体重和脂质含量在整个实验中对应于取样日的脂肪酸和同位素分析。请注意, 在实验期间, 体重的减少和/或脂质含量的降低表明测试机体的适应性降低, 而衍生的数据大大失去了意义。
Isotopologue 检测 isotopologue 分析项目的特殊意义在于对特定脂肪酸中分子离子和所有 isotopologues 的个体检测和定量。例如, 在16:0 的情况下, 离子范围从 270,即,分子离子 M+ (C 骨架主要由12c 原子组成) 到 286 (isotopologue M+16 -碳链完全取代重13C)。图 2A提供了纯16:0、棕榈酸的 SIM-MS 谱。除了分子离子 (M+) 之外, 该化合物中的13C 的自然丰度通过 m+1和 m+2 isotopologues 的存在而变得可检测。相比之下,图 2B显示了本研究中使用的完全标记的棕榈酸 (99 atom% 13C) 的频谱图谱。在这里, 离子 M+16的唯一出现反映了这种综合标记化合物的高纯度。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57110/57110fig2.jpg"> 图 2: 纯 (A) 未标记和 (B) 完全标记为棕榈酸的典型 SIM 扫描.注意 m+1和 m+2 isotopologues 的自然丰度, 除了在未标记的 C 16:0 (A) 中的分子离子 (M+), 但 m+16 isotopologue 在完全标记的脂肪酸中的独占存在 (99个原子% 13C)。<a href="/files/ftp_upload/57110/57110fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
延伸/饱和 vs从头合成 在收获了测试有机体, 油脂提取和衍生化后, 生成的 FAMEs 可以用 GC 和 SIM 质谱分析。通过对 isotopologue 分析数据的检查, 现在可以区分整个标记标记脂肪酸的从头合成和饱和/伸长事件。前者可能发生, 至少部分地, 基于13C 乙酰 CoA 作为被标记的前体的退化的产品通过 beta 氧化。图 3显示了在标签后的第一个取样日, 在H. 大足的 PLFA 分数中从四种主要脂肪酸中提取的 SIM 扫描的典型示例。被标记的标记分子的同化入棕榈酸变得可见由 M+16 isotopologue 的丰盈, 因为它代表完全地被标记的脂肪酸 (图 3A -16:0)。此外, 可以分配饱和 16:0 (图 3B -16:1ω7) 或延伸加饱和 (图 3C -18:1ω9、图 3D 20:4ω6)。因此, 检测大于 M+16的 isotopologues,例如m+17和 M+18通过使用13C2-fragments 演示标记的前体16:0 的链伸长。如果 isotopologues 接近分子离子,即M+1到 M+2 , 则基于13C 的乙酰 CoA 脂肪酸的生物合成的 "从头开始" 是指由0天取样。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57110/57110fig3.jpg"> 图 3: 代表 SIM (选定的离子监测) 对Heteromurus 大足脂肪酸中片段离子的扫描 (PLFA 分数, 标签后1天).(A) 棕榈酸 (16:0) (B) 棕榈油酸 (16:1ω7) (C) 油酸 (18:1ω9) 和 (D) 花生四烯酸 (20:4ω6)。<a href="/files/ftp_upload/57110/57110fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
进一步说明这一点,图 4比较了五个最重标记脂肪酸的初始 isotopologue 模式 (在 PLFAs 和 NLFAs 中进行了分析);比较跳虫物种P. fimata在0天和1天的数据。在1天, C16 和 C18 FAs 中的13C 信号几乎完全基于 M+16分子离子的发生, 其结果完全标记为13C 16:0, 补充到食物。如上所述, M+18 18:0 和18:1ω9的观察到的痕迹建议通过链伸长从16:0 到18:0 引入13C 乙酰 CoA 的一个次要部分。对18:1ω9来说, 这在 PLFA 派系中更为明显。此类13c 乙酰 CoA 来源于标记为13C 16:0 分子的β氧化。合并13C 乙酰 CoA 也发生在伸长步骤从链长度 C18 到 C20, 由 PLFA 样品20:4ω6分配。因此, m+16和 m+18中的13C 富集随着时间的推移显著增加, 直到14天 (图 4)。此外, 与0或1天相比, 此 PLFA 20:4ω6的 M+2在14天增加了。在不同的 isotopologues 中, 13C 分配表示20:4ω6的形成基于合成, 其中包含与 13 c 或在自然 13 c 丰度中标记的乙酰 CoA, 以及整个13C16:0 前体分子的伸长/饱和。相比之下, NLFA 分数 (图 4) 中未显示明显标记为20:4ω6的标签。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57110/57110fig4.jpg"> 图 4: 由 SIM (选定的离子监视) 合并的13C 的 Isotopologue 模式.提出的是离子 m+1到 m+3和 m+16到 m+20五个最沉重标记的 NLFAs 和 PLFAs 从Protaphorura fimata的相对部分, 估计在天0和天1。另外仅为20:4ω6天14数据被提出。P< 0.05 (Dunnett 的测试, 使用0天的数据作为参考)。此数字是在门泽尔et . 中发布的图 5的重印。24<a href="/files/ftp_upload/57110/57110fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers at JoVE.com

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

脂肪酸营养标记方法,即,将脂肪酸同化为整个分子, 并转移到没有或轻微修饰的消费者组织中, 这是由于小土壤无脊椎动物脂肪酸代谢的知识缺口所阻碍的。Isotopologue 分析作为一种宝贵的工具来解开营养的相互作用。

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

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Nanyang Technological University

Research

JoVE Journal

Biochemistry

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

7.9K 次观看.  Humboldt-Universität zu Berlin. 脂肪酸营养标记方法,即,将脂肪酸同化为整个分子, 并转移到没有或轻微修饰的消费者组织中, 这是由于小土壤无脊椎动物脂肪酸代谢的知识缺口所阻碍的。Isotopologue 分析作为一种宝贵的工具来解开营养的相互作用。

视频: Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

脂肪酸鉴定<em&gt;蜡状芽孢杆菌</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

科研

生物化学

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

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

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

用于稳定碳氧同位素分析的牙釉质碳酸盐的取样与预处理

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

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

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

血管紧张蛋白II图解的硝酸分析与鉴定

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

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

脂肪酸¹³C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

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