这项工作得到了国家卫生研究院院长新创新奖DP2OD006466（对K.C.H.）的支持。 作者感谢罗素·蒙兹对方法的实际演示和科学的讨论。

1. 在 2.5 毫升的媒体过夜中培养细菌文化
后冲稀释培养物 1：100 到 250 毫升新鲜介质，并增长到OD 600 的 0.7-0.8。在水中准备6%的硫酸钠（SDS）溶液。
注意：SDS粉末是危险的 - 避免吸入SDS粉末;在鼻子和嘴上戴口罩。
2. 第1天 - 在一天和一夜之间进行莱辛细菌培养
<ol><li>在稀释文化不断发展的同时，在1升烧嘴的热盘子上设置一个开水浴池。水沸腾后，将6毫升6%SDS放入50毫升聚丙烯管中，在每根管子上加入一个小搅拌棒，将管盖固定在手指紧，将管子放入水浴中，并在热板上搅拌至500转/ 时。</li>
	<li>在室温下以 5，000 ×克的速度收获 250 毫升培养物，在室温下 10 分钟，在 3 毫升介质或 1x 磷酸盐缓冲盐水中再补充颗粒。缓慢的移液器细胞悬浮到 50 毫升管中，6% 沸腾 SDS 以解化细胞（最终浓度 4% SDS），而管子则浸入沸水浴中，并将盖子重新封住，以手指紧绷。细胞悬架必须迅速转移到沸腾的SDS一旦恢复。应避免突然的环境变化，因为这可能导致细胞壁结构的错误改变。
</li>
	<li>盖上开水浴池，让细胞煮沸3小时，定期检查水位，必要时重新灌装水浴。3小时后，关闭热板的加热元件，并在500 rpm下持续搅拌过夜。</li>
</ol>3. 第2天 - 酶消化在一天的过程中进行
<ol><li>如果 SDS 在 50 毫升管中沉淀了一夜，则将水浴煮沸 1-2 小时。打开热块至 60 °C。 在 10 mM 特里斯-HCl （pH 7.2） = 0.06% w/v NaCl 中准备 1 毫克/毫升的 Pronase E 库存，并在 60 °C 下激活质氨酶 E 至少 30 分钟。</li>
	<li>使用40万×克的超中心燃料在室温下旋转样品20分钟，以颗粒化大型PG聚合物，从而从其他细胞组件中净化样品。小心去除超细剂，然后在室温超纯水中重新填充每个颗粒。注：悬念体积取决于使用的超中注管的体积：使用至少半路填充管子但不超过管的最大体积的体积。重复离心/清洗，直到水在悬念期间不会形成气泡，指示 SDS 已完全移除（通常为三次洗涤）。如果形成白色沉淀物，请停止清洗颗粒，因为这表明圣餐聚集在一起。结块不是灾难性的，但团块与塑料和玻璃器皿紧密结合，造成大量样品损失。在这种情况下，使用团状的萨库利样本继续执行协议。</li>
	<li>在最后离心/清洗步骤中，将样品重新放在 900 μl 的 10 mM Tris-HCl （pH 7.2） + 0.06% w/v NaCl 中，并转移到 2 毫升管中，以前用小针戳在顶部的孔。在每个样品中加入 100 微克/毫升激活的 Pronase E（100 微克/毫升最终浓度），在 60 °C 下孵育 2 小时。设置不同的热块至100°C。</li>
	<li>通过在每个样品中加入 200μl 的 6% SDS 来阻止 Pronase E 消化，并将样品在 100 °C 热块中煮沸 30 分钟。将不同的热块设置为 37 °C，并在 50 m M 磷酸盐缓冲区 （pH 4.9） 中储存 1 毫克/毫升的穆拉米酶（穆塔诺利辛）。</li>
	<li>与步骤 3.2 一样，使用 400，000 ×克的超中心融合组在室温下旋转样品 20 分钟，并用室温超纯水清洗，直到 SDS 完全去除（通常三次洗涤）。悬念体积取决于使用的超中性管的体积：使用至少半路填充管子但不超过管的最大体积的体积。</li>
	<li>在最后离心/清洗步骤中，在 200 μl 的 50 mM 磷酸钠缓冲器（pH 4.9）中重新悬念样品。此体积可根据样本中的多肽罐量进行调整，并可能取决于物种。如果以前曾发布过关于感兴趣物种的 HPLC 分析报告，则可以根据这些量值来估计此卷（可以在参考7的补充信息中找到 HPLC PG 研究的汇编）。对于其他物种，囊素准备可以执行到此步骤，然后可以添加不同数量的悬念量来复制样本，以确定允许PG留在解决方案中的最小体积（请参阅讨论以进一步估计）。如果样品中含有更多的多肽，增加悬念体积：如果样品几乎没有多肽，则将悬念体积减少到至少 50 μl。</li>
	<li>将样品转移到 1.5 毫升管中，加入 1 毫克/毫升穆拉米达塞，最终浓度为 40 μg/ml。孵化 6-8 小时或在 37 °C 过夜。</li>
</ol>4. 第3天 - UPLC样品的准备在最后一天进行
<ol><li>打开热块至100°C。 在没有 SDS 的情况下煮样品 5 分钟，以阻止穆拉米酶消化。在室温下，在 16，000 ×克处进行 10 分钟的离心取样，然后将超自然（多发性现在可溶性）转移到 13 mm x 100 mm 玻璃管中。尽量恢复超自然，非常接近颗粒，而不会干扰它。</li>
	<li>通过在样品中加入 500 mM 玻酸盐缓冲区 （pH 9） 来调整 pH，最终浓度为 100 mM 玻酸盐缓冲区。玻酸盐缓冲器与减少剂氢化钠兼容。加入1-2粒氢化钠，以减少每个样品，让反应在室温下进行至少30分钟。注意：氢化钠具有高度反应性和危险性 - 避免与皮肤接触（戴手套），避免与眼睛接触（戴安全眼镜）。</li>
	<li>将样品调整到 pH 3-4（多肽等电点为 +3.5），使用 20 μl 增量使用 50% v/v 正磷酸，直到 pH 6，用 pH 指示纸测量，然后使用 2 μl 增量。注意：正磷酸具有腐蚀性和危险性 - 避免接触皮肤（戴手套），避免与眼睛接触（戴安全眼镜）。样品应冒泡，以响应添加矫形磷酸：当样品停止冒泡时，这通常表示已达到 6 的 pH。如果样品中未发生冒泡，这可能表明添加的氢氢钠量太小。在这种情况下，停止降低pH，小心地加入一两粒氢化钠，让反应5-10分钟，然后恢复pH调整。</li>
	<li>通过 0.22μm 注射器过滤器将样品直接过滤到 UPLC 小瓶中。如果沉淀物已经形成，在过滤前，通过火焰多次加热管子。如果样品不会在一天内注入 UPLC 仪器，请在 -20 °C 处冷冻过夜。样品可储存长达一年-80°C。 样品可以通过多次通过火焰解冻。</li>
	<li>将每个样品的 10μl 注入配备 C18 1.7 μm 反向相列的 UPLC 仪器和一个吸收探测器，以监测 202-208 nm。样品按顺序注射，但自动采样器功能允许多达 96 个样品分批处理。使用 50 mM 磷酸钠 （pH 4.35） + 0.4% v/v 钠 azide 溶剂 A， 和 75 mM 磷酸钠 （pH 4.95） + 15% v/v 甲醇溶剂 B. 注： 添加阿齐德钠以补偿甲醇的 205 nm 吸收， 以避免基线漂移。将流量设置为 0.25 毫升/分钟，并在 25 分钟内使用线性梯度，在 30 分钟内实现 100% 溶剂 B 和粘液肽的连续弹性。如果质谱学将用于描述 UPLC 之后的多发性，则收集分数收集器中感兴趣的峰值分数，并使用离心蒸发器干燥分数。</li>
</ol>

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此过程的关键步骤是样品准备第二天的第 3.1 步。如果 SDS 在一夜之间沉淀，或者样品在室温下储存在 4% SDS 中数周，则样品必须重新启动至少 1 小时才能重新溶解 SDS。SDS降水的一个常见原因是使用介质与钾盐，所以钾应避免在媒体，如果可能的话。如代表结果部分所述，将 pH 调整到多兆肽的等电点 （~3.5） 内也至关重要，但不要比 pH 3 低太多，否则材料可能会沉淀。最后，样品的再悬念必须发?...

本文描述的方法的目标是分离完好的细菌细胞壁（sacculi），并消化多肽（PG），以便使用超性能液态色谱（UPLC）提供信息，如多溴肽成分的身份及其浓度，甘油链的平均长度，以及链之间交叉链接所涉及的材料的分数。对于PG生物化学和多溴二苯二甲酸酯物种的详细讨论，有几个优秀的评论，描述了PG结构及其在感染，耐药性，形态形成和生长1-6的作用。用于PG分析的高性能液态色谱（HPLC）最初由格劳纳和施瓦茨在20世纪80年代开发，最近已在米格尔·德·佩德罗和瓦尔德马尔·沃尔默的实验室中得到改进和广泛应用。以前的方法采用氨基酸分析或纸张色谱法，耗时乏味的技术不能产生准确或完整的细胞壁组件评估。
UPLC 分析可轻松地在任何能够访问超中心和 UPLC 的基础研究实验室中实施。我们下面介绍的 UPLC 方法分离了完整的沙库利，从而提供了关于其所有化学物种的全面、定量信息。这种方法可精确量化细菌群中的所有多菌肽，所有在 20 分钟的 UPLC 运行范围内。这种方法的实施只涉及基本的实验室技能，没有对材料进行重大财政投资。为了执行这种方法的步骤，研究人员只需要熟练地吹笛，准备缓冲器和酶，并调整pH，使其能够进入广泛的科学学科。本协议中使用的酶的选择取决于所分析的细菌种类：这里描述的协议对 大肠杆菌是有用的，并且通常被发现足以将沙丘利与其他格拉姆阴性生物隔离开来。建议在将这种方法应用于革兰阳性细菌时与文献协商：在这些物种中，囊菌净化历来比较困难。特别是，这种方法可能必须改变酶的选择和消化时间的长度，以适应较厚的墙壁和附属聚合物，如革兰氏阳性细菌的铁酸。本协议中的第一种酶将外膜脂蛋白（如布劳恩的脂蛋白或Lpp）附着在肽上，从而从细胞壁中释放除C-终端二（或三）肽外的所有肽。此步骤在检查肠杆菌时是必要的，但许多其他 Gram 阴性细菌没有 Lpp 等价物，因此可以跳过此步骤。第二种酶在多肽的穆拉米酸成分后特别切开，溶解形成木荷肽物种的脱糖亚单位。为了准确评估 PG 的结构，应小心消化沙库利，以防止跨桥或肽茎的任何其他部分的。
虽然HPLC对来自100多个菌株的多肽类的化学成分进行了分析，但与普遍性保护委员会技术没有进行任何分析。此外，先前的工作仅从细菌领域的一小部分对多肽进行描述，部分受 HPLC 的吞吐量的限制。因此，将这种方法传播给尽可能多的研究人员，并在普遍支持下平台上实施，对于推动对大部分尚未分类的细菌物种进行生理研究至关重要。

<table border="0" cellpadding="0" cellspacing="0" width="640">
	<colgroup>
		<col />
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Pronase E</td>
			<td style="width:115px;">Amresco</td>
			<td style="width:119px;">E629</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mutanolysin from Streptomyces</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">M9901</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium borohydride (NaBH4)&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">452882</td>
			<td>Sodium borohydride is highly reactive and dangerous to handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orthophosphoric acid&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">79607</td>
			<td>Orthophosphoric acid is corrosive and dangerous to handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric acid</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">31146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2002</td>
			<td>Sodium azide is a poison</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium tetraborate</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">221732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex 0.22 &#956;m syringe filters</td>
			<td style="width:115px;">Fisher</td>
			<td style="width:119px;">SLGVR04NL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH strips (pH range 0-6)</td>
			<td style="width:115px;">Fisher</td>
			<td style="width:119px;">M95863</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml polypropylene Falcon tubes</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">21008-951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">13 mm x 100 mm glass tubes</td>
			<td style="width:115px;">Kimble Chase</td>
			<td style="width:119px;">60CM13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">12 mm x 32 mm screw neck glass recovery vial</td>
			<td style="width:115px;">Waters</td>
			<td style="width:119px;">186000327C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Sodium Dodecyl Sulfate</td>
			<td style="width:115px;">Ambion</td>
			<td style="width:119px;">AM9820</td>
			<td>SDS powder is hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;"></td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Instrumentation</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Waters Acquity UPLC H-Class system, including:</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Acquity UPLC H-Class Sample Manager FTN</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Acquity UPLC H-Class Quaternary Solvent Manager</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Acquity UPLC BEH C18 1.7 &#181;m column</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquity UPLC PDA Detector</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waters Fraction Collector III</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Acquity UPLC 30 cm Column Heater/Cooler</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography

细菌细胞壁对于确定细胞在生长和分裂过程中的形状至关重要，在面对各种大气中的涡轮压力时保持细胞的机械完整性。在细菌王国的不同形状和大小中，细胞壁由多肽组成，这是一个由短肽交叉连接的糖链的巨分子网络。Peptidoglycan对细菌生理学的核心重要性是其作为抗生素靶点的使用的基础，并促使遗传、结构和细胞生物学研究如何在生长和分裂过程中牢固地组装它。尽管如此，仍需进行广泛的调查，以充分描述多肽合成中的关键酶活动和细菌细胞壁的化学成分。高性能液相色谱 （HPLC） 是量化在各种环境和遗传条件下生长的细菌壁化学成分差异的有力分析方法，但其吞吐量往往有限。在这里，我们提出了一个简单的程序，通过HPLC和超性能液态色谱（UPLC）对多肽菌进行生物分析的细菌细胞壁的隔离和制备，这是HPLC的延伸，利用泵提供高达15，000 psi的超高压，而HPLC的超高压为6，000 psi。结合这里介绍的细菌细胞壁的制备，低容量的样品喷油器、采样率高、样品量小、运行时间短的检测器，将使大多数生物实验室能够获得超中心和 UPLC，实现多肽类成分和基本细菌细胞生物学的新发现。

使用 图 1中概述的程序，最终样本应包含至少 200 μl 的清晰溶液，这些溶液已直接过滤到 UPLC 小瓶中（第 4.4 步）。在细菌样本中分离各种多发性肽的 UPLC 取决于它们在液体移动相和柱的静止阶段之间的相对溶解性。反向阶段C18柱提供强烈的疏水基质，根据疏水性和大小8分离多溴二苯甲酸酯物种：极性、低分子量的单体先阐明，后是极性、分子量较高的寡聚物（图2）。...

Watch this Scientific Journal Video about Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography at JoVE.com

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

细菌细胞壁由多肽组成，多肽交叉连接的糖链大分子网络。超性能液态色谱为多肽类成分的新发现提供了高分辨率和高吞吐量。我们提出了一个隔离细胞壁（sacculi）的程序，以及随后通过 UPLC 进行分析的准备。

通过超性能液相色谱对细菌细胞壁进行隔离和制备，进行组成分析

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

isolation-preparation-bacterial-cell-walls-for-compositional-analysis

Research

JoVE Journal

Chemistry

16.0K 次观看.  Stanford University. 细菌细胞壁由多肽组成，多肽交叉连接的糖链大分子网络。超性能液态色谱为多肽类成分的新发现提供了高分辨率和高吞吐量。我们提出了一个隔离细胞壁（sacculi）的程序，以及随后通过 UPLC 进行分析的准备。

视频: Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

科研

化学

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

使用气相色谱怀孕期间分析脂肪酸组成的变化在大鼠肝组织

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Synthesis and Catalytic Performance of Gold Intercalated in the Walls of Mesoporous Silica

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties of the materials were investigated. We used a one pot sol-gel approach to intercalate gold nano particles in the walls of mesoporous silica. To start with the synthesis, P123 was used as template to form micelles. Then TESPTS was used as a surface modification agent to intercalate gold nano particles. Following this process, TEOS was added in as a silica source which underwent a polymerization process in acid environment. After hydrothermal processing and calcination, the final product was acquired. Several techniques were utilized to characterize the porosity, morphology and structure of the gold intercalated mesoporous silica. The results showed a stable structure of mesoporous silica after gold intercalation. Through the oxidation of benzyl alcohol as a benchmark reaction, the GMS materials showed high selectivity and recyclability.

Here, we present a protocol with a sol-gel process to synthesize gold intercalated in the walls of mesoporous materials (GMS), which is confirmed to possess a mesoporous matrix with gold intercalated in the walls imparting great stability and recyclability.

合成和金插在介孔二氧化硅的墙壁催化性能

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties ...

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

柱后衍生用反应流高效液相色谱柱

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

简捷方法对硫甙的提取与分析用高压液相色谱法（HPLC）

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

用于跟踪纳米粒子自组装的液体细胞透射电子显微镜

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...