Name: quantitative and qualitative method for sphingomyelin by lc-ms using two stable isotopically labeled sphingomyelin species
Uploaded: 2018-05-07T13:00:00
Description: Watch this Scientific Journal Video about Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species at JoVE.com

这项工作得到了日本教育、文化、体育、科学和技术部 (KAKENHI) 对 K.H. (#15K01691)、许贤 (#15K08625)、肯塔基州 (#26461532) 的研究补助金的支助, 并获得了教育部关于顽固性疾病项目的研究补助金。健康、劳动和福利 (肯塔基州 #201510032A)。我们感谢 Edanz 集团 (www.edanzediting.com/ac) 编辑这份手稿的草稿。

使用前请查阅所有相关的材料安全数据表 (MSDS)。戴上手套, 以减少皮肤衍生 SM 的样品污染。本议定书适用于在鹰的最低基本培养基中生长的 HeLa 细胞, 辅以10% 胎牛血清 (血清)、2毫米l-谷氨酰胺、1000 U/升青霉素和100毫克/升链霉素。
1. 脂质样品的制备
注: 重要的是所有的玻璃器皿, 包括含聚四氟乙烯衬里螺丝帽的试管是无洗涤剂的。
<ol><li>用布莱 & ...

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在目前的定性方法中, 我们获得了 SPC 的3产品离子和一个N-酰基团。正确分配 SPC 和N-酰基基团是至关重要的。为此, 应该指出的是, 其他含有氯化磷的分子也可以被检测为 MS3产品离子。Diacyl 磷脂酰胆碱 (pc) 和缩醛磷脂-pc 在哺乳动物细胞中大量存在, 其疏水性与 SM 相似。因此, diacyl-pc 和缩醛磷脂-与同位素 (通常为13 C) 的 pc 可以在理论上与 SM 同时检测。在实验?...

鞘磷脂 (SM) 是哺乳动物细胞中常见的鞘脂。SM 是合成 intracellularly1和目前作为其他鞘脂类的前兆, 如 sphingosine-1-phosphate 和神经酰胺, 这在免疫细胞贩运和皮肤屏障稳态的关键作用, 分别为2, 3。因此, 对 SM 代谢的精确分析对于阐明鞘脂类的生理和病理作用具有重要意义。
SM 由神经酰胺和氯化磷组成, 它与神经酰胺的 1-羟基基团相连, 它由一个鞘和一个N-酰基团组成。鞘和N-酰基基团中的碳和双键数的变化导致神经酰胺 (和 SM) 种类的数量。LC-ESI/ms 的最新进展使 SM4、5的数量和质量分析得以实现。在定性分析中, 通过分配 LCB 的产品离子谱, 确定了 SM sphingoid LCB 的碳和双键的数量。但是, 由于未报告相应的产品离子, 所以未直接获得n-酰基基团的结构信息, 因此, 通过对前驱离子之间的微分分析, 导出了n酰基团, 并在正负离子模式下与 LCB 对应的产品离子4,5。在本报告中, 我们提出了一种用三重四极和四极线性离子阱质谱法在 MS3模式中同时检测 LCB 和N酰基团的产物离子的方法, 这有助于精确的结构对每个 SM 物种的推测6。
基质在生物样品中所引起的离子抑制 (或增强) 效应阻碍了 LC-ESI-质谱分析中的精确量化, 因此, 在相同矩阵中, 为所有感兴趣的解析度构造校正曲线是可取的。的生物样本。然而, 这一策略是不可行的, 因为几乎不可能准备所有 SM 物种的生物样本, 特别是在综合分析。因此, 在生物样品中, 采用有代表性的 SM 种来构造标定曲线, 确定定量范围是切实可行的。我们用两个 isotopically 标记的 SM 种来构造一个定标曲线;一个用于内部标准, 另一个用于标准化合物。我们检测出少量的 isotopically 标记的 SM 品种作为标准化合物在生物样品中被刺, 并且成功地获得了校正曲线和定量范围6。

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PBS</td>
			<td style="width:131px;">ThermoFisher</td>
			<td style="width:119px;">10010023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">100 mm tissue culture dish</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">3020-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Cell scraper</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">9000-220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Siliconized 2.0 mL tube</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:119px;">02-681-321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Test tube</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">TST SCR 16-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teflon-lined screw cap</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">9998CAP415-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Disposable glass tube</td>
			<td style="width:131px;">IWAKI</td>
			<td style="width:119px;">9832-1310</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">CAPCELL PAK C18 ACR 3 &#181;m 1.5 mm I.D. x 100 mm</td>
			<td style="width:131px;">Shiseido</td>
			<td style="width:119px;">92223</td>
			<td>Guard cartridge is inserted into cartridge holder, and linked to C18 column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">CAPCELL C18 MGII S-3 2.0 mm x 10 mm GUARD CARTRIDGE</td>
			<td style="width:131px;">Shiseido</td>
			<td style="width:119px;">12197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Cartridge holder</td>
			<td style="width:131px;">Shiseido</td>
			<td style="width:119px;">12415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Acetonitrile</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">018-19853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">2-Propanol (Isopropyl Alcohol)</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">161-09163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Methanol</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">134-14523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Formic acid</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">066-00466</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">28% Ammonia water</td>
			<td style="width:131px;">Wako</td>
			<td style="width:119px;">016-03146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sonicator (bath type)</td>
			<td style="width:131px;">SHARP</td>
			<td style="width:119px;">UT-206H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Vortex mixer for glass test tube</td>
			<td style="width:131px;">TAITEC</td>
			<td style="width:119px;">Mix-EVR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1.4 mL glass vial</td>
			<td style="width:131px;">Tomsic</td>
			<td style="width:119px;">500-1982</td>
			<td>Samples are stored in 1.4 mL glass vial sealed with screw cap and 8 mm septum at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">8 mm septum</td>
			<td style="width:131px;">Tomsic</td>
			<td style="width:119px;">200-3322</td>
			<td>When samples are analyzed, screw caps are replaced with screw caps with slit septum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Screw cap for 1.4 mm glass vial</td>
			<td style="width:131px;">Tomsic</td>
			<td style="width:119px;">500-2762</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Screw cap with slit septum</td>
			<td style="width:131px;">Shimadzu GLC</td>
			<td style="width:119px;">GLCTV-803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PVDF 0.22 &#181;m filter</td>
			<td style="width:131px;">Millipore</td>
			<td style="width:119px;">SLGVR04NL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">Triple quadrupole and quadrupole linear ion trap mass spectrometry</td>
			<td style="width:131px;">SCIEX</td>
			<td style="width:119px;">QTRAP4500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">The software for data acquisition and analysis of product ion spectra</td>
			<td style="width:131px;">SCIEX</td>
			<td style="width:119px;">Analyst</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">The software for data integration in quantitative analysis</td>
			<td style="width:131px;">SCIEX</td>
			<td style="width:119px;">MultiQuant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HPLC system</td>
			<td style="width:131px;">Shimadzu</td>
			<td style="width:119px;">Nexera</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Glass bottle</td>
			<td style="width:131px;">Sansyo</td>
			<td style="width:119px;">85-0002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">d18:1/24:0 sphingomyelin</td>
			<td style="width:131px;">Avanti Polar Lipids</td>
			<td style="width:119px;">860592P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sphingosylphosphorylcholine</td>
			<td style="width:131px;">Merck</td>
			<td style="width:119px;">567735</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:131px;">ThermoFisher&#160;</td>
			<td style="width:119px;">26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">L-glutamine</td>
			<td style="width:131px;">ThermoFisher&#160;</td>
			<td style="width:119px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Penicillin and streptomycin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eagle&#8217;s minimum essential medium</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:119px;">M4655</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative and qualitative method for sphingomyelin by lc-ms using two stable isotopically labeled sphingomyelin species

本文提出了用液相色谱-电喷雾电离-串联质谱 (LC-鞘磷脂) 定性和定量分析 (SM) 的方法。SM 是由氯化磷和神经酰胺组成的一种常见鞘脂, 分别为亲水性和疏水性成分。由于 sphingoid 长链基 (LCB) 和神经酰胺中的一个N酰基团的变化, 许多 SM 物种存在于哺乳动物细胞中。在本报告中, 我们展示了一种估算 LCB 中碳和双键的数量和一个基于其相应的产品离子的N-酰基基团 (ms3) 实验的方法。此外, 我们提出了一个定量的分析方法的 sm 使用两个稳定的 isotopically 标记 sm 物种, 这有助于确定的范围内使用的 sm 定量。本方法将有助于在生物样品和化妆品等工业产品中表征各种 SM 品种。

化学合成的 d18:1/24:0 sm ( 图 1A) 和 d18:1/24:0 从 HeLa 细胞提取的脂质样本 (图 1B) 通过使用 [m + HCOO] 和 [m CH3] 的 LC-ESI-MS3进行分析-作为第一和第二前体离子分别。请注意, demethylated-sphingosylphosphorylcholine (SPC) (m/z 449) 的频谱强度大于 SM N-酰基基团 (m/z 378)。?...

Watch this Scientific Journal Video about Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species at JoVE.com

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

用两个稳定 Isotopically 标记鞘磷脂种鞘磷脂的定量和定性方法

在这里, 我们提出了一个协议, 以量化和合格的每个鞘磷脂物种使用多反应监测和 ms/毫秒/ms 模式, 分别。

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

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Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

毛细管电聚焦法对蛋白质及其亚型的高灵敏度和定量检测

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

通过并行反应监测进行无处不在的链分析

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

强大的单颗粒冷冻电子显微镜 （cryo-EM） 处理工作流程，具有冷冻 SPARC、RELION 和 Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

使用定量流式细胞术分析荧光标记蛋白与人工磷脂微泡的相互作用

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...