这项工作由 NSF 植物基因组研究计划赠款 IOS-1238812 和生物学 IOS-1400818 博士后研究员资助。这项工作还由孟山都研究生奖学金给凯瑟琳 a. 萨蒙斯的资助。富布赖特非洲研究员学者计划 (2017-2018) 感谢授予 Nokwanda p 麻昆加的资金。我们非常感谢从杰西卡 Prenni 的 nanospray 来源和科罗拉多州立大学蛋白质组学和新陈代谢设施的捐赠。

1. 对 Nanospray 源的改质
<ol><li>使用改进的 nanospray 源进行叶喷雾 MS。由于叶喷雾 MS 不需要射流元件, 所以通过从源中移除 LC 探头来修改源。</li>
	<li>组装叶喷雾 MS 丝, 将应用电压的植物组织与适当的针插入源。将引脚焊接到绝缘导线的一端;将钳位焊在导线的另一端。 注: 夹子 (鳄鱼夹子类型) 可能或可能没有牙。对于小组织, 无牙钳是首选。一个可选的弹性臂与钳位可以添加到 nanospray 源, 以协助定位的植物组织。请注意, 本协议具体描述了如何在混合四极离子阱质量分析仪 ms 系统上执行叶喷雾 ms (见材料表);但是, 其他 MS 系统可能会被改变以执行这项技术6。耦合叶喷雾 MS 与便携式质谱仪实时化学分析可以在现场进行, 不需要运输植物材料到实验室19,20。</li>
	<li>将防静电地板垫放在电源下面的地板上, 以减少在使用高压时从电源上产生的电放电。</li>
</ol>2. 叶喷雾 ms 系统的研制
<ol><li>如果系统最近被使用, 允许它冷却到触摸和删除任何替代源和扫锥。附上 nanospray 叶喷雾 MS 源。</li>
	<li>创建具有适当电离参数设置的调谐文件, 如下所示: 鞘、辅助和扫气为 0;喷雾电压为 2-5 伏;毛细管温度到 150-250 °c;和 S 透镜 RF 水平到50。将调谐文件保存为所需的参数8、13。优化电压和温度的最佳电离的组织和化合物 (s) 的兴趣。 注: 良好的起始点为4伏和200°c。</li>
	<li>制作一个方法文件, 包括叶喷雾 ms 调谐文件: 正、负全 ms;7万的决议;1 x 106的 AGC 目标;最大值为200毫秒;和所需的扫描范围m/z。或者, 只使用1极性。</li>
</ol>3. 仪器、溶剂和植物组织的制备
注意:始终戴手套, 不要使用徒手处理过的植物组织。否则, 诸如聚乙二醇等污染物离子将主宰光谱。
<ol><li>将植物组织的分析带到与 MS 系统相同的房间, 以便快速取样。</li>
	<li>对于没有天然尖尖的植物组织, 在玻璃滑梯上使用刀片来切割锥形点 (图 2)。根据仪器灵敏度、组织类型和感兴趣的化合物 (例如, 长约5毫米的幼tortuosum叶) 确定分析所需的组织数量。<ol>
<li>tortuosum叶在10周后萌发成薄条, 各有一个锥形端形成一个点。</li>
	</ol>
</li>
	<li>使用镊子轻轻地选择将被夹紧的末端的植物组织。用镊子握住纸巾, 小心地将其转移到夹钳上。 注意: 如果电压为 on, 请勿触摸仪器电源。</li>
	<li>用夹钳调整挠性手臂和导线, 使组织与 ms 入口线保持一致, 这样植物组织与 ms 离子入口之间的距离为三重四极 (如TSQ) 和线性圈闭四极 (LTQ) 和10。-50 毫米离子阱质量分析仪 (例如, orbitrap)8。<ol>
<li>将导线的另一端插入源。如果第一次尝试产生低信号强度, 移动植物组织接近离子入口 (参考讨论为优选)。</li>
	</ol>
</li>
	<li>加载方法文件;命名数据文件并设置文件存储位置。然后通过单击 "播放" 打开 MS 系统, 然后单击 "开始" 开始获取数据。</li>
	<li>应用溶剂 (如甲醇), 使用吸管与凝胶加载尖端, 以最大的距离之间的手和高电压, 以保护用户。 注: 所需溶剂量取决于组织的大小、干燥度和质地, 一般为 2-20 µL tortuosum叶不需要添加任何溶剂。在电压不动的情况下, 小心地应用溶剂, 不要接触仪器电源。使用经酸洗的 LC-MS 级溶剂和玻璃器皿, 无洗涤剂。在某些组织中, 由于植物组织的自然含水量, 在不添加溶剂的情况下可以观察到信号。然而, 一个更大的信号强度和减少的 S/N 通常是通过应用溶剂的组织来实现。</li>
	<li>获取数据, 只要信号持续存在, 或者直到收集到足够的光谱, 通常为30六十年代。如果需要, 应用额外的溶剂保持较高的信号强度, 在较长的时间。停止数据获取并暂停 MS 系统。</li>
	<li>取下纸巾, 用100% 甲醇和无绒布擦拭钳洗。根据供应商的规格, 在大约 1-2 小时的采集后, 清洁 ms 离子入口。同时, 在不同组织类型的分析之间清除 MS 离子入口。</li>
</ol>4. 数据质量评估
<ol><li>打开数据文件, 目视检查基峰值质量 chronogram。检查信号强度是 ~ 1.0 x 107到 5.0 x 108。如果信号较低, 将组织移近离子入口。如果较高, MS 系统的前端将变得肮脏, 所以将组织进一步从离子入口。</li>
	<li>根据所产生的质谱中的感兴趣离子的存在或缺失, 改变参数。 注意: 协议可以在这里暂停。</li>
</ol>5. 串联质量碎片
<ol><li>确定哪些离子对串联质量碎片有兴趣 (ms/毫秒);1.0 x 105的质谱信号对于 ms/ms 的离子选择是足够的。</li>
	<li>制作一个新的方法文件, 其中包含的m/z的收录列表到4个小数位。点击全局列表和包含。在PRM 的属性下, 选择碎片能量 [例如, 30-50 的规范化碰撞能量是一个良好的起始范围] 和其他 ms/ms 参数。<ol>
<li>为了获得 mesembrine 生物碱的 ms/ms 数据, 片段的以下离子, 276.1583米/z, 290.1742米/z, 292.1897米/z, 在。 注意: ms 或 ms 数据采集可以在 ms 或以后的时间内立即执行。同样的组织可以在完整 ms 后保持钳位, 并且可以重用以获得 ms/ms 数据。但是, 如果 respraying 没有提供足够的信号, 请使用新的组织。</li>
	</ol>
</li>
	<li>加载 ms/毫秒方法文件和命名数据文件。打开 MS 系统, 开始获取数据, 必要时添加溶剂。当收集到足够的光谱, 通常在30六十年代之后, 停止获取。</li>
	<li>在分配片段离子时, 收集许多不同能量的碎片。 注: 由于叶喷雾 MS 缺乏色谱分离, 碎片谱可能含有许多离子, 而在不同能量下的分解将有助于使其清晰。</li>
</ol>6. 精确质量和串联质量碎片的假定识别
<ol><li>通过引用来自公共可用的代谢物数据库 (如 Metlin21、人类代谢物数据库22、大众银行23、脂质图24、美国国家研究所) 的精确质量测量来做出假定的标识。标准和技术 MS 搜索25, 尊重植物化学物质26, 或大亚湾27。</li>
	<li>由于这些数据库并非详尽无遗, 因此需要对具有化学特征的植物种类进行额外的文献综述。</li>
	<li>当 ms 或 ms 信息可用或文献时, 将碎片离子从叶喷雾 ms/ms 与上述数据库匹配。或者, 使用手动解释 ms/毫秒片段离子或由直接注入或 LC ms/ms 执行的真实标准的碎片。</li>
</ol>7. 数据分析
<ol><li>使用 msConvert 工具从 Proteowizard28将 MS 原始文件转换为 mzXML 文件。</li>
	<li>使用 R 中实现的 XCMS 软件包进行峰值拾取。采用直接输液处理方法对叶喷雾进行 MS 分析。 注意: 用于数据处理的注释好的脚本可以在 https://github.com/HegemanLab/Leaf-Spray-Code 中找到。</li>
	<li>为了获得半定量测量, 会计的实验变异性, 标准化的强度, 每一个代谢物的总离子电流 (TIC), 因为叶喷雾 MS 信号强度可能会有所不同, 部分原因是在定位的细微变化叶在来源和叶子的形状和大小的区别。</li>
	<li>或者, 使用供应商提供的软件进行数据分析或 MZmine2 (在 http://mzmine.github.io/中找到)29。</li>
</ol>

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作者没有什么可透露的。

该协议的成功使用依赖于植物种类、组织类型和目标化合物的各种步骤的优化。协议中描述的参数提供了一个良好的起点。以下实验决定需要做和测试: 是否使用 (1) 切开或未切割的组织和 (2) 溶剂或无溶剂, (3) 什么溶剂使用和在什么容量, (4) 从离子入口的组织距离应该是什么, (5) 电压振幅。优化的目标是找出产生连续信号的条件, 持续至少三十年代到几分钟。条件应提供足够的和可重现的信号强度, 这是需要执行准确的质量和 ms/毫秒测量。高信号强度是通过成功和可靠的喷雾离子从组织。喷雾质量取决于指向离子入口的组织尖端的锋利, 组织的尖端和离子入口之间的距离, 以及所应用的电压。成功的喷雾高度依赖于组织尖端的锐度, 在某些情况下, 组织应该被切割成尖尖。特别是, 在切口组织尖端的锥形形状角锐度的细微变化对由此产生的电离质量有显著影响, 从而产生7的信号强度。如果组织已经指出, 那么没有切割是必要的, 就像草叶片或柳叶刀形状的叶子5,13的情况下。
离子信号可以被抑制和不稳定, 当等离子放电发生从组织由于被放置太接近离子入口或电压过高。需要适当的组织定位和电压的选择, 以确保样品之间的稳定和一致的喷雾。MS 离子入口的组织距离也会影响信号的质量和产量。一般来说, 较小的组织样本应放置在靠近入口, 虽然小规模的组织可能不会导致低信号强度, 如果尖端是非常尖锐或化合物高度集中。通过比较沿 z 轴的几个位置位置, 可以对光谱的强度和一致性进行优化。在 x 和 y 轴中, 植物组织与 MS 离子入口对齐, 这是一个合适的电离的关键。然而, 如果植物组织不合理地接近离子入口, 这可能需要更加频繁的清洁离子光学和 MS 系统的前端。
在组织顶部应用的溶剂体积可从 0-50µL, 视含水量和组织样品的大小而定。如果组织的含水量极高且被切割, 如肉质Sceletium的情况, 则不能添加溶剂。然而, 至少在一个应用中使用至少 5-10 µL 溶剂是比较典型的。加入溶剂是必要的, 当使用一个干组织或新鲜组织与低含水量, 以促进喷雾。如果在一大块植物材料上使用少量的溶剂, 它很可能会被吸收而不产生足够的喷雾。反之, 如果使用量太高, 化合物可能被稀释或适当的 desolvation 不会轻易和有效地发生。手动吹打溶剂的另一种选择是通过注射器泵不断地将溶剂应用到组织中, 这样, 当化合物从植物材料10中耗尽时, 所观察到的信号就会衰减为时间的函数。应尝试各种类型的溶剂, 并与所得到的光谱进行比较, 以验证所用化合物 (s) 的数量和一致性的改善。添加溶剂不仅能产生喷雾, 而且还能为不同化合物的萃取提供选择性。对不同极性的有机溶剂 (甲醇、二氯甲烷、hexanes、乙腈、氯仿和丙酮) 进行了比较, 结果表明, 花生籽8的光谱中存在明显不同的离子。一般来说, 甲醇是一个很好的第一溶剂选择, 因为它已被证明对许多植物组织和多种化学物质, 包括氨基酸, 生物碱, 黄酮醇, 碳水化合物, 有机酸, 脂肪酸, 磷脂8的良好工作。100% 水在未修剪的植物组织中的应用通常不会很好地工作, 但可以通过添加盐10来改善。在许多情况下, 除了质子离子的化合物, 其他丰富的加合物检测, 如钠和钾加合物。当盐添加到溶剂中时, 这些盐加合物的存在就更普遍了, 而且是有利的。例如, 添加钠和钾离子到应用溶剂10, 观察到杨树中酚苷的敏感性和选择性增加。
叶片喷雾 MS 技术的两大局限性是 (1) 低动态范围和 (2) 定量的挑战。通常, 只有最丰富的化合物被电离和检测的技术。由于在缺乏色谱分离的情况下发生的离子抑制而导致的电离效率降低, 这是一个富含代谢物的问题。为了规避这一限制, 扫描范围可以调整为只专注于感兴趣的m-/z范围。然而, 如果不采用色谱法分离和浓缩, 低丰度化合物仍可能无法检测到。与从萃取物中提取的化合物的典型定量不同, 内部标准在叶喷雾前不能适当地混入植物材料中. 通过放置已知的半定量测量和相对浓度获得浓度的标准溶液在组织表面, 然后允许它干燥之前, 叶喷雾 MS 分析8,9,31。例如, 用定量标准加法法计算内部标准的代表性离子与感兴趣的离子的比值, 以确定相对数量32。用校正曲线估计相对浓度。利用这种方法, 可以将不同苷的比值与内部标准进行比较, rebaudioside D, 然后通过叶33计算出特定苷的相对浓度. 或者, 一个更准确的定量是可能的 isotopically 标记标准的复利, 虽然商业可用性可能是一个挑战。使用代谢标记植物组织也可以提高定量与此方法34。
鉴于传统的 LC-ms/ms 需要广泛的样品制备和色谱分离, 其他分析方法往往是可取的。叶喷雾 MS 是一种直接的化学分析技术, 可以很容易地应用, 并提供简单, 准确, 准确和快速代谢物检测, 半定量。为此, 我们研究了叶喷雾 MS 对tortuosum的化学含量进行监测的适宜性, 为 chemotaxonomic 在生物化学基础上鉴别Sceletium属的种类提供依据。签名。该植物的几种解剖特性使其成为叶片喷雾 MS 的理想试样。它是一种肉质的, 含有大量的水, 这是有利的, 因为喷雾可以产生没有溶剂的应用。Sceletium叶含有 idioblasts (膀胱样细胞)15 , 可作为贮存储备, 在那里可以积累专门的代谢物。叶喷雾 MS 是一种体内分析技术, 以快速的方式描述植物组织。一般技术适用于许多植物种类、组织类型和化合物类别。捕获植物化合物信息的技术对于了解植物的主要和专门的代谢, 以供人类使用健康、营养、农业和能源35非常感兴趣。

植物含有多种化学性质的小分子。MS 是一种强有力的分析植物化合物的技术, 因为它能为代谢产物的检测和鉴定提供高灵敏度和特异性的元素成分1。最常见的是, ms 是在溶剂提取的样品, 这是分离的色谱之前, ms 分析1。然而, 使用液相色谱 (LC) 需要冗长的分析时间, 往往与广泛的样品准备1。相反, 直接化学分析的完整组织, 绕过色谱是一个非常迅速的技术, 需要最小的样品准备2。因此, 在色谱步骤可以被放弃的情况下, 直接的化学分析可以是非常有利的。
典型的 LC MS 为自然产品和新陈代谢研究依靠长的散装提取干燥或冷冻植物材料包含多组织和细胞类型3。或者, 直接化学分析, 如 MS 检测植物组织的代谢物, 可以隔离细胞类型和避免准备工件4。叶喷雾 ms, 也称为组织喷雾5,6, 是一个直接的环境电离 MS 技术, 这要求基本上没有样品准备5,7。叶喷雾 ms 与纸面喷雾 ms 密切相关, 这是一种具有电喷雾电离特性的环境电离技术, 可以检测沉积在纸7上的分析物。尽管名称, 叶喷雾 MS 是适用于各种类型的植物组织, 而不仅仅是叶子, 并已证明在水果, 种子, 根, 花卉组织, 块茎, 其中包括6,8,9, 10,11,12。该技术促进了内源植物化学物质直接从植物材料电离到质谱仪中检测8。叶喷雾 MS 还可以提供关于植物中不同组织类型的化学物质的空间分布的信息13。当叶喷雾 ms 与溶剂萃取和 LC ms 相比, 结果表明, 叶喷雾 ms 允许快速检测的表面代谢产物, 从独特的细胞类型, 如毛13。图 1说明了叶喷雾 MS 实验设置。直接电喷雾电离只在少量的源修改后发生。高压通过金属钳被应用到植物组织中, 产生一喷雾高电荷的水滴形成一个泰勒锥, 将离子传送到 MS 的离子入口. 电喷雾电离发生从植物的天然液体或溶剂李波爆炸到植物表面。一个尖尖的组织促进了电喷雾, 可以自然地发生或通过切割创建。
叶喷雾 MS 是一种快速的方法, 定性和半定量分析的完整的植物组织, 已发现效用广泛的应用。例如, 该技术被用来检测内源化合物, 以区分相关物种, 甚至评估在不同条件下生长的同一物种的变化。以前的研究通过测定紫珠中的代谢产物 (紫L) 表明了这种方法。12和美国人参(西洋参)6. 在后一种例子中, 可以在润湿生人参后发现人参皂甙、氨基酸和寡糖。野生和栽培西洋参被区分从块茎切片6。人参块茎的完整性保存后, 叶喷雾 MS, 允许随后的形态学和显微检查6。此外, 还可以检测植物样品中的外源化合物。在9水果和蔬菜的果皮或果肉上发现了一些杀虫剂 (脒、二苯胺、唑、linuron 和噻)。虽然这些研究和许多其他已经表明了叶喷雾 MS 的用途, 为各种特定目的, 一个详细的协议没有以前报告。
在这里, 协议描述不会集中于对特定组织或化合物的方法的优化。相反, Sceletium tortuosum (L.) n.e. (Aizoaceae) 中的 mesembrine 生物碱的检测被用作一个例子, 讨论在为物种、组织或化合物 (s) 第一次。tortuosum是南非半干旱 Karroo 地区特有的肉质植物。一种传统医学的 San 和同属的民族, 它被用于食欲和口渴抑制以及它的精神和镇痛作用14,15。目前, 标准提取物用于精神和神经心理疾病的治疗16,17。感兴趣的主要化合物包括生物碱 mesembrine 及其衍生物, 其中许多也见于相关的Sceletium物种15。tortuosum的野生和栽培种群都有 mesembrine 生物碱的浓度变化, 从而提出了质量控制挑战18。一种快速检测 mesembrine 生物碱的方法, 如叶喷雾 MS, 可用于监测Sceletium产品。由于以前没有对叶喷雾 MS 技术进行详细的可视化实验协议, 我们将用S. tortuosum的例子说明该方法, 并描述了以下内容: nanospray 源的修改、植物组织的选择和制备, 数据的获取, 结果的解释, 以及 MS 参数的优化。

<table border="0" cellpadding="0" cellspacing="0" width="880">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Conn Pin</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td style="width:119px;">WM2563CT-ND</td>
			<td style="width:385px;">pin will insert into Thermo Scientific&#160; source to provide voltage&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">small clamp</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td>314-1018-ND</td>
			<td>CLIP MICRO ALLIGATOR COPPER 5A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">large clamp</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td>290-1951-ND</td>
			<td>ALLIGATOR CLIP NARROW NICKLE 5A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat shrink</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td style="width:119px;">Q2Z1-KIT-ND</td>
			<td>to cover soldering joints&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">NSI source Nanospray Ion Source</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;">NA</td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">Q Exactive- hybrid quadrupole Orbitrap</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;">NA</td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Tune Software</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Xcalibur Software</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Plant of interest - S. tortousum</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

leaf spray mass spectrometry: a rapid ambient ionization technique to directly assess metabolites from plant tissues

植物产生数以千计的小分子, 它们的化学性质多种多样。质谱 (MS) 是一种有效的分析植物代谢产物的技术, 因为它提供了高灵敏度和特异性的分子量。叶喷雾 MS 是一种环境电离技术, 植物组织用于直接化学分析通过电喷雾, 消除色谱的过程。这种取样代谢物的方法允许从完整的植物组织同时检测出多种化学类, 从而尽量减少所需的样品准备量。当使用高分辨率, 准确的质量 ms, 叶喷雾 ms 促进快速检测代谢物的兴趣。还可以用这种技术收集串联质量碎片数据, 以促进复合识别。准确的质量测量和碎片的结合有利于确认复合恒等式。叶喷雾 MS 技术只需要小的修改 nanospray 电离源, 是一个有用的工具, 进一步扩大的能力, 质谱仪。本文对南非传统药用植物Sceletium tortuosum (Aizoaceae) 的鲜叶组织进行了分析;用叶喷雾质谱检测了大量 mesembrine 生物碱。

在发芽后10周, 用叶喷雾质谱分析了新鲜收集的温室tortuosum叶。用叶喷雾 MS 检测tortuosum叶中代谢产物的实验工作流程如图 2所示。选择一叶, 切成带锥形端的薄条, 形成点, 并夹持叶状喷涂 MS 线钳装置。植物组织被安置了 ~ 30 毫米从离子入口并且与 x 和 y 轴一致。在tortuosum叶的叶喷雾剂启动后, 检测到没有任何溶剂的离子。Sceletium家族的植物肉质植物具有高水分含量的厚叶, 在没有溶剂的外部应用的情况下, 叶喷雾 MS 能使电喷雾电离。然而, 最典型的, 植物组织需要一个溶剂应用来检测离子。
大量的 chronogram 和大量的质谱在三十年代的收购之后产生, 在正电离模式下 (图 3)。chronogram 代表一个成功的叶喷雾 MS 实验, 在整个采集期间保持稳定的信号。在本研究中, 用叶喷雾剂检测了九 mesembrine 生物碱, 并根据先前特征化合物 (表 1)30的质子离子的确切质量进行了假定鉴定。在高分辨率、精确质量 (HRAM) 质谱仪上执行叶喷雾 MS 是有利的, 这使得可以解决细微差别的肿块。例如, m/z在262.1794 可以被认定识别为 dihydrojoubertiamine 而不是 mesembrenone m (demethyl 氢), 这将是在m/z 262.1443。
叶组织保持夹紧的串联质量 (ms/毫秒) 碎片的几个离子的利益 (表 1)。图 4显示了来自叶喷雾 ms、276.1583米/z、290.1742米/z和 292.1897米/z的 ms/毫秒碎片谱的三例。假定的身份被质量碎片验证, 并确认以前确定的片段30。质子离子 276.1583米/z被确定为两个不同异构体的 mesembrine m (demethyl) 和另两个异构体的 mesembrenone m 由于存在的诊断片段为每个异构体 (表 1)。
mesembrine 和 mesembrine (双氢) 均通过叶片喷雾质谱和认定, 并通过质量分裂来检测。mesembrine 在 290.1742米/z的质子离子是光谱中最丰富的离子之一;因此, 它可能是一个可能的候选物作为未来研究的生物标志物。tortuosum的两种主要生物碱是 mesembrine 和 mesembrenone, 它们都能通过叶喷雾质谱检测得到。在Sceletium和衍生产品的植物材料的监测中, 一些由叶喷雾 MS 检测的化合物可能是有用的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57949/57949fig1v2.jpg"> 图 1: 设置的叶喷雾 MS 图.叶喷雾 MS 是一种代谢物分析方法, 允许快速取样完整的植物组织。该图显示了用 kV 高电压的叶喷雾 MS 和夹具, 并将溶剂应用于植物组织的选择。叶喷雾 ms 促进电喷雾电离直接从植物组织进入 MS 入口。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57949/57949fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57949/57949fig2.jpg"> 图 2: 叶喷雾 MS 实验工作流.选定的植物组织, 一Sceletium tortuosum叶, 被切开, 然后转移与钳被夹紧, 然后安置在离子入口前面在数据承购之前。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57949/57949fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57949/57949fig3v2.jpg"> 图 3: Sceletium tortuosum的代谢产物的叶喷雾 MS 与正电离.(a) 此面板显示了叶喷雾 MS 总离子计数 (TIC) 质量 chronogram。对于每个峰值, 顶部的数字是时间 (分钟), 底部是m/z。(B) 本小组显示Sceletium tortuosum正质谱的叶喷雾 MS 代谢物剖面。插入显示 260-295 m/z。准确的质量被报告到4小数位, 误差 < 6 ppm。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57949/57949fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57949/57949fig4v2.jpg"> 图 4: 叶喷雾 MS 正离子串联质谱从Sceletium tortuosum切叶.这些面板显示的串联质谱 (ms/毫秒) 收集的正电离模式与叶喷雾 MS. 生物碱的鉴定是由精确的质量和质量碎片组成的: (A) mesembrine 和 mesembrenone-m异构体, (B) mesembrine 和 (C) mesembrine (二氢)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57949/57949fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>复合</td>
			<td>分子公式</td>
			<td>测量的 Monoisotopic 质量m/z [m + H]</td>
			<td>ppm 错误</td>
			<td>碎片</td>
		</tr>
<tr>
<td>Dihydrojoubertiamine</td>
			<td>C16H23NO2
</td>
			<td>262.1794</td>
			<td>5。0</td>
			<td>N/A</td>
		</tr>
<tr>
<td>Mesembrenone (O-demethyl-)</td>
			<td>C16H19NO3
</td>
			<td>274.1431</td>
			<td>4。5</td>
			<td>N/A</td>
		</tr>
<tr>
<td>Mesembrine (O-demethyl-)</td>
			<td>C16H21NO3
</td>
			<td>276.1583</td>
			<td>5。9</td>
			<td>121, 152, 195, 201, 218, 219, 258</td>
		</tr>
<tr>
<td>Mesembrine (n-demethyl)</td>
			<td>C16H21NO3
</td>
			<td>276.1583</td>
			<td>5。9</td>
			<td>109, 121, 138, 189, 201, 218, 247</td>
		</tr>
<tr>
<td>Mesembrenone (O-demethyl-双氢-)</td>
			<td>C16H21NO3
</td>
			<td>276.1583</td>
			<td>5。9</td>
			<td>124, 205, 218, 227, 245</td>
		</tr>
<tr>
<td>Mesembrenone (n-demethyl)</td>
			<td>C16H21NO3
</td>
			<td>276.1583</td>
			<td>5。9</td>
			<td>120, 151, 210, 229, 241</td>
		</tr>
<tr>
<td>Mesembrenone</td>
			<td>C17H21NO3
</td>
			<td>288.1587</td>
			<td>4。3</td>
			<td>124, 151, 191, 199, 226, 230, 257, 270</td>
		</tr>
<tr>
<td>Mesembrine</td>
			<td>C17H23NO3
</td>
			<td>290.1742</td>
			<td>4。9</td>
			<td>110, 121, 134, 152, 201, 215, 219, 232, 233, 241, 259, 260, 272</td>
		</tr>
<tr>
<td>Mesembrine (双氢)</td>
			<td>C17H25NO3
</td>
			<td>292.1897</td>
			<td>5。2</td>
			<td>151, 177, 201, 217, 243, 259, 274</td>
		</tr>
</tbody></table>表 1: 假定的标识Sceletium tortuosummesembrine 生物碱的叶喷雾质谱.本表报道了Sceletium tortuosum mesembrine 生物碱的正电离准确质量和片段离子。

Watch this Scientific Journal Video about Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues at JoVE.com

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

叶喷雾质谱法是一种直接的化学分析技术, 它能最大限度地减少样品的制备, 消除色谱, 从而快速检测植物组织中的小分子。

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

leaf-spray-mass-spectrometry-rapid-ambient-ionization-technique-to

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.5K 次观看.  University of Minnesota. 叶喷雾质谱法是一种直接的化学分析技术, 它能最大限度地减少样品的制备, 消除色谱, 从而快速检测植物组织中的小分子。

视频: Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

激光显微切割RNA测序实验设计:从玉米叶片发展的经验分析

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

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

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

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

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

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

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

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

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

探头电喷电镀质谱的样品制备

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

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

从高梁叶组织中分离出希石，用于自上而下质量光谱分析潜在的表观遗传标记

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...