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 & 戴尔方法从细胞匀浆中提取总脂质分数7<ol>
<li>从10厘米的组织培养皿中取出培养 (或有条件的) 培养基, 用6毫升的冰冷 PBS 冲洗两次。</li>
		<li>用 P1000 吸管将1毫升冰冷 PBS 加入10厘米的组织培养皿中。用细胞刮刀收割细胞并收集成2.0 毫升的渗塑料管。</li>
		<li>离心后 (1000 x 克, 5 分钟, 4 摄氏度), 用 P1000 吸管去除上清液。</li>
		<li>加入1毫升甲醇。涡流管简要地和油脂实验在 200 W 为5分钟在浴式 sonicator。 注: 调节浴式 sonicator 中的水位, 使细胞颗粒有效地匀质。</li>
		<li>将吸管中的细胞匀浆转化成试管 (13 毫米 x 100 毫米), 用聚四氟乙烯衬里螺钉盖。</li>
		<li>加入1毫升甲醇, 1 毫升氯仿, 0.8 毫升的双蒸馏水, 50 µL 的10µmol/升的内部标准 d18:1/(D31)-16:0 SM 进入每个试管。</li>
		<li>涡流试管在室温下用力5分钟。 注: 此时, 形成一个单相 (氯仿/甲醇/水 = 1/2/0.8 (v/v/v))。</li>
		<li>加入1毫升氯仿和1.0 毫升的双蒸馏水。</li>
		<li>涡流管以 2500 rpm 的力度在室温下5分钟。 注: 此时, 水 (上) 相和有机 (下) 相分离。</li>
		<li>离心后 (2150 x 克, 5 分钟, 25 °c), 收集和转移到一次性玻璃管 (初始较低阶段) 的低相。 注: 使用巴斯德吸管和安全吸管过滤器收集下一阶段。将吸管的尖端插入下一阶段, 挤压 "E" 阀旁边的小灯泡, 以便在吸管的尖端内传递上相和界面绒毛, 然后将下相虹吸到吸管中。</li>
		<li>在试管中加入2毫升的氯仿, 在室温下以 2500 rpm 为5分钟, 充分地与上相和界面绒毛混合。</li>
		<li>离心后 (2150 x 克, 5 分钟, 25 °c), 收集和转移修改后的低相进入一次性玻璃管与初始较低阶段所述的步骤1.1.10。</li>
		<li>将玻璃管置于氮气流下, 并在所收集的较低相中完全去除有机溶剂。</li>
		<li>用500µL 甲醇或乙醇重建样品, 用0.02 µm 过滤器滤液, 在-20 摄氏度的玻璃瓶中贮存。</li>
	</ol>
</li>
	<li>构造标定曲线的样品制备及验证方法<ol>
<li>将标准化合物 (d18:1/(D5)-10 SM) 的0.1、0.5、1、50、9或18:1 µmol/L 的50µL 添加到带有聚四氟乙烯衬里螺钉帽的试管中。</li>
		<li>在每个试管中加入细胞匀浆, 并将甲醇加入1毫升。 注: 细胞匀浆作为基质的数量根据每个实验而变化。如果从10厘米培养皿中的细胞中提取的 SM 种类进行例行分析, 则每个试管中的细胞匀浆量应接近10厘米培养皿中的细胞。</li>
		<li>提取总脂分数, 如步骤 1.1. 6-1. 1.14 所述。</li>
		<li>准备质量检查 (QC) 样品以在步骤 1.2. 1-1. 2.3 中验证方法为望见。用不同浓度的标准化合物 (d18:1/(D9)-18:1 SM) 制备三个 QC 样品: 一个3x 以内的标准曲线 (qc-低, qc L) 的下限, 一个靠近中心 (qc 中间, qc M), 和一个附近的上部边界标准曲线 (qc-高, qc H)。</li>
	</ol>
</li>
</ol>2. 经 LC-ESI/ms 分析
<ol><li>移动阶段的准备<ol>
<li>混合溶剂 (乙腈/甲醇/ddH2O = 2/2/1 (v/v/v) 的水相和异丙醇的有机相) 在玻璃瓶与聚四氟乙烯衬里螺钉帽和油脂实验5分钟的浴式 sonicator。</li>
		<li>添加甲酸 (最终浓度26.4 毫摩尔/升) 和 NH 4 OH (14.9 毫摩尔/升) 进入每个移动阶段.</li>
	</ol></li>
	<li>基于 LC-ESI-MS3的 SM 质量分析<ol>
<li>激活高效液相色谱 (HPLC) 系统, 将进口管放入含有流动相的玻璃瓶中, 并清除液相色谱线。链接 C18高效液相色谱柱 (1.5 毫米 x 100 毫米长度, 粒度3.0 µm) 到高效液相色谱系统, 将温度保持在柱式烘箱的50摄氏度, 并将该柱与水流动相在100µL/分钟. 将样品放入 autosampl 的样品架中二。</li>
		<li>设置三极和四极线性离子阱质谱系统的参数, 用于 MS3分析, 如表 1和表 2中所列, 以及感兴趣的 SM 种类的第一和第二前驱离子。<ol>
<li>双击 "软件" 图标进行数据采集, 双击 "硬件配置", 选择 "LC+QTRAP4500+Valve", 然后单击 "激活配置文件"。</li>
			<li>通过单击 "新建子项目" 和 "确定" 创建新的子文件夹。</li>
			<li>单击 "文件" 选项卡, 选择 "新建", 然后选择 "获取方法" 和 "确定"。单击 "质量规范" 图标和 "ms" 选项卡. 选择扫描类型为 ' ms/毫秒/ms (MS3) ', 扫描率为 1万 Da/秒, 极性为 ' 负 ', 和 ' 持续时间 ' 作为全时分析 (min)。将 "开始 (da)" 和 "停止 (da)" 的m/z设置为要扫描的范围。设置感兴趣的目标 SM 种类的第一和第二前体离子的m-/z 。 注: 要分析多个 sm 物种, 请突出显示 "-MS3" 图标, 右键单击, 选择 "复制此实验", 然后将其他 SM 物种的第一和第二前体离子的m-/z 。</li>
			<li>选择 "高级 MS" 选项卡, 并按如下方式设置每个参数: 扫描模式为 "剖面", 步长为 0.12 (Da), 分辨率 Q1 和 Q3 ' 单位 ' 和 ' 点亮 ', 分别为强度阈值为 0, 设置时间为 50 (毫秒), 在质量范围之间暂停为1.5 毫秒, 选择 "动态填充时间, Q3 进入屏障为 8 V, 激发时间为25毫秒。</li>
			<li>单击 "MS" 选项卡中的 "编辑参数" 按钮, 然后选择 "源/气体" 选项卡. 设置窗帘气体、碰撞气体、ionspray 电压、温度、离子源 gas1 和 gas2 的参数, 如表 1所示。然后选择 "复合" 选项卡. 设置分割电位、入口电位、碰撞能量、励磁能量和碰撞能量的参数, 如表 2所示。</li>
			<li>突出显示 "集成瓦尔科阀" 并选择 "步骤 0" 的位置名称作为 A, 并将 "B" 设置为总时间5.0 分钟的位置。还设置 ' A ' 为总时间70.0 分钟的位置。</li>
			<li>突出显示 "日本岛津 LC 系统"。选择 "泵" 选项卡, 并将泵浦模式设置为二进制流量, 总流量为0.28 毫升/分钟, 最大压力限制为20.0 兆帕。选择 "Autosampler" 选项卡, 并将漂洗量设置为200µL, 针冲程为52毫米, 冲洗速度为35µL/秒, 取样速度为5.0 µL/秒, 清洗时间为25.0 分钟, 冲洗时间为5秒, 冲洗方式为 "前后吸"。<ol>
<li>另外, 使冷却器单位, 设置冷却器温度为4°c 和控制小瓶针冲程作为52毫米. 选择 "烤箱" 标签, 使烤箱和设置烤箱温度和最高温度分别为50摄氏度和85摄氏度。</li>
				<li>选择 "控制器" 选项卡并选中 "通电"。选择 "时间程序" 选项卡, 并设置溶剂梯度如下: 溶剂 a/B 在100/0 的比率为5分钟, 程序线性改变到 80/20 4 分钟, 到 35/65 50 分钟, 和到 25/75 1 分钟之后, 将它们保持在 25/75, 10 分钟, 然后线性到 100/0 4 分钟。然后保存该方法。</li>
			</ol>
</li>
		</ol>
</li>
		<li>创建批处理文件<ol>
<li>双击 "生成购置批次" 图标, 单击 "添加集", "添加示例", 设置新示例的数量, 然后单击 "确定"。</li>
			<li>单击 "方法编辑器" 按钮, 然后选择要使用的方法。如果在一个批处理文件中使用了多个方法, 请选中 "使用多个方法" 框, 并为每个示例选择获取方法。重命名 "样本名称", 为 "瓶子位置" 设置适当的编号, 并放入注入量。然后保存批处理文件。</li>
		</ol>
</li>
		<li>通过 LC-ESI-MS3分析获得 SM 类感兴趣的 ms3产品离子谱<ol>
<li>在批处理文件中选择 "提交" 选项卡, 突出显示要分析的样本行, 然后单击 "提交" 按钮。</li>
			<li>单击 "查看 Quene" 图标, "平衡" 图标, 选择要使用的采集方法, 将时间设置为1分钟, 然后单击 "确定"。</li>
			<li>通过单击相应的图标, 停用 "用于调整的备用工具"。然后通过单击 "开始示例" 图标来执行批处理序列。</li>
		</ol>
</li>
		<li>通过比较产品离子谱的质量与电荷比 (m/z) 和 sphingoid LCB 的确切质量和N-酰基团, 分配 SM 的每个 MS3产品离子谱。根据相应的产品离子, 确定 sphingoid LCB 和N酰基团中的碳和双键的数量。<ol>
<li>请确认选择了相应的子项目文件夹, 然后双击 "打开数据文件" 图标, 然后选择要分析的示例。在色谱中拖动每个目标 SM 品种的峰值, 然后双击。在拖动区域内获得的 MS3产品离子谱将被提出。</li>
		</ol>
</li>
	</ol></li>
	<li>用 LC-质谱法定量分析 SM 的含量<ol>
<li>按照步骤2.1 和2.2.1 中的说明设置移动相和 HPLC 柱。</li>
		<li>将三重四极和四极线性离子阱质谱系统的参数设置为多反应监测 (MRM) 分析, 如表 1和表 2中所列。<ol>
<li>执行步骤2.2.2.1 和2.2.2.2 中所述的过程。</li>
			<li>单击 "文件" 选项卡, 选择 "新建", 然后选择 "获取方法" 和 "确定"。单击 "质量规范" 图标和 "MS" 选项卡. 选择扫描类型为 "MRM", 极性为 "正数"。将 "工期" 设置为要分析的整个时间。分别将 [m + H]+和184作为目标 SM 种类的 "Q1 质量 (da)" 和 "Q3 质量 (da)" 的m/z 。设置 "时间" 为10毫秒和 ' ID ' 作为目标 SM 物种的名称。</li>
			<li>选择 "高级 MS" 选项卡, 然后将每个参数设置为: 两个分辨率 Q1 和 Q3 为 "单位", 强度阈值为 0, 将时间设置为 0 (毫秒), 并在质量范围之间暂停为5毫秒。</li>
			<li>单击 "MS" 选项卡中的 "编辑参数" 按钮, 然后选择 "源/气体" 选项卡. 将窗帘气体、碰撞气体、ionspray 电压、温度、离子源 gas1 和 gas2 的参数列在表 1中。然后选择 "复合" 选项卡. 将分割电位、入口电位、碰撞能量和碰撞细胞出口电位等参数列在表 2中。</li>
			<li>按照步骤2.2.2.6 中的说明设置阀门。</li>
			<li>按照步骤2.2.2.7 中的说明设置 LC 条件。然后保存该方法。</li>
		</ol>
</li>
		<li>按照步骤2.2.3 中的说明创建批处理文件。</li>
		<li>按照步骤2.2.4 中的描述, 通过 LC-ESI-ms/毫秒分析获得每个 SM 物种的 MRM 数据。</li>
		<li>利用软件进行数据集成处理 MRM 数据, 获取各 SM 品种提取的离子色谱峰面积数据。<ol>
<li>双击软件图标进行数据集成在定量分析中, 单击 "编辑" 选项卡并选择 "用户集成默认值"。将高斯平滑宽度设置为1.0 点, 然后单击 "确定"。单击 "编辑" 选项卡, 然后选择 "新建结果表"。选择要集成的示例, 单击箭头按钮, 然后单击 "下一步"。选择 "创建新方法", 设置方法的名称, 然后单击 "下一步"。单击 "下一步" 并选中 d18:1/(D31)-16:0 SM 的复选框, 单击 "下一步", 然后单击 "完成"。</li>
			<li>单击 "显示峰值审阅", 并确认色谱峰被适当识别。应注意的是, d18:1/(D31)-16:0 sm 的保留时间通常小于0.3 分钟, 与 34-1 SM (通常由 d18:1/16:0 sm) 之一相比。</li>
		</ol>
</li>
		<li>根据每个 sm 物种的峰值与 d18:1/(D31)-16:0 sm 内部标准的比值来量化每个 sm 物种。<ol>
<li>单击 "文件" 选项卡, 选择 "导出", 选择 "结果表", 确认格式为 "MultiQuant", 列为 "导出所有列", 行为 "导出除那些显式隐藏的所有行", 然后单击 "确定"。</li>
			<li>在 Excel 软件中打开导出的文件。通过区域 (d18:1/(D31)-16:0 sm) 正常化目标 SM 物种的区域。然后乘以注入样本中的理论量, 计算注射样品中靶 SM 种类的数量。</li>
		</ol>
</li>
	</ol></li>
	<li>构造标准曲线<ol>
<li>获取 d18:1/(d9)-18:1 sm 和 d18:1/(d31)-16:0 sm 的 MRM 数据, 这些示例用于构造标准曲线的 LC-ESI-ms/毫秒分析, 如步骤2.3 中所述. 1-2. 3.4。</li>
		<li>获取 d18:1/(d9)-18:1 sm 和 d18:1/(d31)-16:0 sm (如步骤2.3.5 中所述) 的峰值区域的数据。</li>
		<li>按照步骤2.3.6 中所述, 计算注入的样本中的 d18:1/(D9)-18:1 SM 的数量。</li>
		<li>通过将 X 轴和 Y 轴设置为 d18:1/(D9)-18:1 SM 的标称量和计算量来构造趋势线, 并获取趋势线公式。</li>
	</ol></li>
	<li>使用 Excel 软件验证量化方法<ol>
<li>要验证该方法, 请通过在三天内至少1次分析每个 qc 示例, 量化 d18:1/(d9)-18:1 sm 和 d18:1/(d31)-16:0 sm 在 qc 样品中的数量, 并重复至少3天, 如步骤 2.3. 1-2. 3.4 所述。</li>
		<li>获取峰值区域数据并计算在注入的样本中的 d18:1/(D9)-18:1 SM 的数量, 如步骤2.3.5 和2.3.6 中所述。</li>
		<li>根据所得到的校准曲线, 计算注入样品中的 d18:1/(D9)-18:1 SM 的量。</li>
		<li>精度评定<ol>
<li>计算 d18:1/(D9)-18:1 SM 在日内和天间使用 Excel 软件的数据集上获得的2.5.3 量的平均值和标准偏差。</li>
			<li>在步骤2.5.4.1 中获得的平均值除以标准偏差, 并表示为%。</li>
		</ol>
</li>
		<li>准确性评估<ol>
<li>计算每个数据的准确性, 如下所示: [(在步骤2.5.3 中获得的计算量)。/(标称金额)-1] x 100 (%)</li>
			<li>计算日内和日间数据集的精度绝对值的平均值。</li>
		</ol>
</li>
	</ol></li>
</ol>

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C., Kelly, S., Wang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipidomics:+high-throughput,+structure-specific,+and+quantitative+analysis+of+sphingolipids+by+liquid+chromatography+tandem+mass+spectrometry.">Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.</a> Methods. 36 (2), 207-224 (2005).</li><li>Houjou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+selective+identification+of+molecular+species+in+phosphatidylcholine+and+sphingomyelin+by+conditional+neutral+loss+scanning+and+MS3.">Rapid and selective identification of molecular species in phosphatidylcholine and sphingomyelin by conditional neutral loss scanning and MS3.</a> Rapid Commun Mass Spectrom. 18 (24), 3123-3130 (2004).</li><li>Hama, K., Fujiwara, Y., Tabata, H., Takahashi, H., Yokoyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Quantitation+Using+Two+Stable+Isotopically+Labeled+Species+and+Direct+Detection+of+N-Acyl+Moiety+of+Sphingomyelin.">Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of N-Acyl Moiety of Sphingomyelin.</a> Lipids. , (2017).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37, 911-917 (1959).</li><li>Gu, H., Liu, G., Wang, J., Aubry, A. F., Arnold, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+the+correct+weighting+factors+for+linear+and+quadratic+calibration+curves+with+least-squares+regression+algorithm+in+bioanalytical+LC-MS/MS+assays+and+impacts+of+using+incorrect+weighting+factors+on+curve+stability,+data+quality,+and+assay+performance.">Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance.</a> Anal Chem. 86 (18), 8959-8966 (2014).</li><li>Folch, J., Lees, M., Sloane Stanley, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues.">A simple method for the isolation and purification of total lipides from animal tissues.</a> J Biol Chem. 226 (1), 497-509 (1957).</li><li>Thomas, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ozone-induced+dissociation:+elucidation+of+double+bond+position+within+mass-selected+lipid+ions.">Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions.</a> Anal Chem. 80 (1), 303-311 (2008).</li><li>Baba, T., Campbell, J. L., Le Blanc, J. C., Baker, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+sphingomyelin+characterization+using+electron+impact+excitation+of+ions+from+organics+and+mass+spectrometry.">In-depth sphingomyelin characterization using electron impact excitation of ions from organics and mass spectrometry.</a> J Lipid Res. 57 (5), 858-867 (2016).</li><li>Ryan, E., Nguyen, C. Q. N., Shiea, C., Reid, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+Structural+Characterization+of+Sphingolipids+via+193+nm+Ultraviolet+Photodissociation+and+Ultra+High+Resolution+Tandem+Mass+Spectrometry.">Detailed Structural Characterization of Sphingolipids via 193 nm Ultraviolet Photodissociation and Ultra High Resolution Tandem Mass Spectrometry.</a> J Am Soc Mass Spectrom. , (2017).</li><li>Pham, H. T., Ly, T., Trevitt, A. J., Mitchell, T. W., Blanksby, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+complex+lipid+isomers+by+radical-directed+dissociation+mass+spectrometry.">Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry.</a> Anal Chem. 84 (17), 7525-7532 (2012).</li></ol>

作者声明他们没有利益冲突。

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。在实验中, 缩醛磷脂的MS 3 产品离子在 SM 分析中同时被观察到。正确选择 SM 的 MS3产品离子有助于了解 SPC的频谱强度大于 SM N-酰基基团 (图 1和B)。相比之下, 脂肪酰基团的强度大于 (或几乎相同) 的 demethylated lysoplasmalogen-PC (数据没有显示)。
在目前的定量方法中, 精确地准备样本来构造标定曲线和验证方法是至关重要的。此外, 应适当使用加权因子来构造标定曲线;特别是在低浓度下, 改善曲线拟合是有益的。用不同的加权因子对标定曲线进行了比较。使用加权因子 = 1/x2与使用加权因子 = 1 或 1/x (图 2B) 的比较, 明显改善了低浓度曲线拟合。精度和准确度的值应在15% 以内。如果 QC L 的浓度与标准曲线的下边界相同 (定量的下限), 则该值应在 20%8内。
本研究采用了布莱 & 戴尔法提取培养细胞的总脂质分数。其他脂质提取方法, 如 Folch 方法也很有用9。根据样品的用量和/或性质, 用适当的方法提取脂质分数是很重要的。在每个注入中同时分析的 SM 种类的数量不应该太大, 以防止软件超载的数据采集。预定的 MRM 将有助于同时量化多个 SM 物种。
我们目前使用 MS3分析的方法对于推测 LCB 和N-酰基基团的碳和双键的数量没有额外的设备是有用的。但是, 不能获得其他结构信息, 如双键位置、异构体 (cis或反式) 和形状 (直或分枝)。有必要使用更高的能量来获得产品离子及其结构信息使用附加仪器10,11,12,13。与N-酰基基团对应的产品离子的分子公式为 [RCO2]-不含氮气的离子。目前尚不清楚碰撞引起的离解是如何进行的。
对于 ms3分析, 必须调整 ms3碎片 (ExT) 的碰撞能量 (CE) 和激发时间参数。较高的 CE 将导致多余的碎片, 并降低 demethylated SM 的产品离子的强度作为2的nd前体离子。此外, 碎片模式明显依赖于 ExT。在实验之前, 最好是通过将合成 sm 溶解在移动相中来确定 demethylated sm、SPC 和N酰基团的产品离子强度最大化的 CE 和 ExT 的适当条件。
我们使用 d18:1/(d9)-18:1 sm 和 d18:1/(d31)-16:0 sm 作为两个 isotopically 标记 sm 物种, 以构造校准曲线。电离的效率可以根据 SM 种类和流动相的条件而变化。因此, 如果感兴趣的 sm 的数量是有限的, 这是可取的准备 isotopically 标记的 SM 物种的利益, 以更准确的定量。
根据前体离子和与 demethylated SPC 对应的产物离子, 确定了 SM 结构。此外, 在样本矩阵中, 由于感兴趣的目标化合物大量存在, 因此有时会妨碍精确确定存在样本矩阵中定量的下限。
本研究是有用的, 以估计的碳和双键在 LCB 和一个N酰基团的基础上, 其相应的产品离子在 MS3实验没有额外的仪器。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。本方法将有助于在各种生物样品和工业产品中表征各种独特的 SM 品种。

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。

<table><tbody><tr><td>PBS</td><td>ThermoFisher</td><td>10010023</td><td></td></tr><tr><td>100 mm tissue culture dish</td><td>IWAKI</td><td>3020-100</td><td></td></tr><tr><td>Cell scraper</td><td>IWAKI</td><td>9000-220</td><td></td></tr><tr><td>Siliconized 2.0 mL tube</td><td>Fisher Scientific</td><td>02-681-321</td><td></td></tr><tr><td>Test tube</td><td>IWAKI</td><td>TST SCR 16-100</td><td></td></tr><tr><td>Teflon-lined screw cap</td><td>IWAKI</td><td>9998CAP415-15</td><td></td></tr><tr><td>Disposable glass tube</td><td>IWAKI</td><td>9832-1310</td><td></td></tr><tr><td>CAPCELL PAK C18 ACR 3 &amp;micro;m 1.5 mm I.D. x 100 mm</td><td>Shiseido</td><td>92223</td><td>Guard cartridge is inserted into cartridge holder, and linked to C18 column</td></tr><tr><td>CAPCELL C18 MGII S-3 2.0 mm x 10 mm GUARD CARTRIDGE</td><td>Shiseido</td><td>12197</td><td></td></tr><tr><td>Cartridge holder</td><td>Shiseido</td><td>12415</td><td></td></tr><tr><td>Acetonitrile</td><td>Wako</td><td>018-19853</td><td></td></tr><tr><td>2-Propanol (Isopropyl Alcohol)</td><td>Wako</td><td>161-09163</td><td></td></tr><tr><td>Methanol</td><td>Wako</td><td>134-14523</td><td></td></tr><tr><td>Formic acid</td><td>Wako</td><td>066-00466</td><td></td></tr><tr><td>28% Ammonia water</td><td>Wako</td><td>016-03146</td><td></td></tr><tr><td>Sonicator (bath type)</td><td>SHARP</td><td>UT-206H</td><td></td></tr><tr><td>Vortex mixer for glass test tube</td><td>TAITEC</td><td>Mix-EVR</td><td></td></tr><tr><td>1.4 mL glass vial</td><td>Tomsic</td><td>500-1982</td><td>Samples are stored in 1.4 mL glass vial sealed with screw cap and 8 mm septum at -20&amp;deg;C</td></tr><tr><td>8 mm septum</td><td>Tomsic</td><td>200-3322</td><td>When samples are analyzed, screw caps are replaced with screw caps with slit septum</td></tr><tr><td>Screw cap for 1.4 mm glass vial</td><td>Tomsic</td><td>500-2762</td><td></td></tr><tr><td>Screw cap with slit septum</td><td>Shimadzu GLC</td><td>GLCTV-803</td><td></td></tr><tr><td>PVDF 0.22 &amp;micro;m filter</td><td>Millipore</td><td>SLGVR04NL</td><td></td></tr><tr><td>Triple quadrupole and quadrupole linear ion trap mass spectrometry</td><td>SCIEX</td><td>QTRAP4500</td><td></td></tr><tr><td>The software for data acquisition and analysis of product ion spectra</td><td>SCIEX</td><td>Analyst</td><td></td></tr><tr><td>The software for data integration in quantitative analysis</td><td>SCIEX</td><td>MultiQuant</td><td></td></tr><tr><td>HPLC system</td><td>Shimadzu</td><td>Nexera</td><td></td></tr><tr><td>Glass bottle</td><td>Sansyo</td><td>85-0002</td><td></td></tr><tr><td>d18:1/24:0 sphingomyelin</td><td>Avanti Polar Lipids</td><td>860592P</td><td></td></tr><tr><td>Sphingosylphosphorylcholine</td><td>Merck</td><td>567735</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher&amp;nbsp;</td><td>26140079</td><td></td></tr><tr><td>L-glutamine</td><td>ThermoFisher&amp;nbsp;</td><td>25030081</td><td></td></tr><tr><td>Penicillin and streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Eagle&amp;rsquo;s minimum essential medium</td><td>Sigma</td><td>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)。此外, 与 [M-胆碱-CH3 (sphingosine-1-phosphate)] 对应的频谱也有助于分配 SM 的 LCB.我们还确认, demethylated spc (m/z 449) 的频谱主要是由 d18:1 SPC 在我们的 MS3分析 (图 1C) 的条件下产生的。
使用双 isotopically 标记 SM 种的校准曲线显示在图 2A中。通过将 1/x2作为加权因子来获得趋势线。剩余分析的结果显示在图 2B中。请注意, 通过应用 1/x2作为加权因子, 接近定量 (本研究中的 0.1 pmol) 的剩余值较小。
得到的校准曲线和验证结果的参数显示在表 3中。精度和准确度的值在15% 以内, 表明本研究符合标准, 是使用 LC MS8的定量方法。
在 HeLa 细胞中每个 SM 物种的数量都显示在表 4中。HeLa 细胞生长在培养培养基中, 含有10% 的血清和收获。d18:1/16:0 sm 和 d18:1/24:1 sm 是最主要的和第二个最丰富的 sm 物种的结构确定, 并由54% 和14% 的总 SM, 分别
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57293/57293fig1.jpg"> 图 1.MS3在 HeLa 细胞中 SM 的特定m-/z信号的光谱.本文展示了从 HeLa 细胞 (B) 提取的脂质样品中化学合成的 d18:1/24:0 SM (A) 和 d18:1/24:0 sm 的3谱。结果由哈马et进行了修改。6注意到 SPC 的频谱强度大于 SM 的N-酰基基团. 通过使用相同的 m-/z ([m + HCOO]-, m, 化学合成的 d18:1 SPC (C) 中显示的 MS3谱。/z 499) 作为第一和第二前体离子。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57293/57293fig1large.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/57293/57293fig2.jpg"> 图 2.使用两个 isotopically 标记的 sm 种类的 sm 校准曲线.(A) 使用双 isotopically 标记 SM 种类 (d18:1/(d9)-18:1 sm 和 d18:1/(d31)-16:0 sm) 的校准曲线。校准曲线是使用加权因子 = 1/x2构造的。验证的结果列在表 3中。(B) 用于评估构造校准曲线的优点的剩余分析。使用加权因子 = 1/x2的校准曲线中的残差小于使用加权因子 = 1/x 或1的值. 特别是在较低的 d18:1/(D9)-18:1 SM.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57293/57293fig2large.jpg" target="_blank">请单击此处查看更大版本的此图.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td colspan="6">TurboIonSpray 设置</td>
		</tr>
<tr>
<td>模式</td>
			<td>窗帘气 (psi)</td>
			<td>碰撞气体</td>
			<td>离子喷涂电压 (V)</td>
			<td>温度 (°c)</td>
			<td>离子源气体 1 (psi)</td>
			<td>离子源气体 2 (psi)</td>
		</tr>
<tr>
<td>MS3
</td>
			<td>40</td>
			<td>高</td>
			<td>-4500</td>
			<td>200</td>
			<td>40</td>
			<td>80</td>
		</tr>
<tr>
<td>Mrm</td>
			<td>40</td>
			<td>10</td>
			<td>5500</td>
			<td>300</td>
			<td>40</td>
			<td>80</td>
		</tr>
</tbody></table>表1。电喷雾离子源的条件用于定性和定量分析.此表是从哈马et改编而来的。6
<table fo:font-size="6" fo:keep-together.within-page="1"><tbody>
<tr>
<td>模式</td>
			<td>极性</td>
			<td>前体离子 (Q1)</td>
			<td>产品离子或第二前驱离子 (Q3)</td>
			<td>分割电位 (V)</td>
			<td>入口潜力 (V)</td>
			<td>碰撞能量 (V)</td>
			<td>碰撞细胞出口电位 (V)</td>
			<td>碰撞能量传播 (V)</td>
			<td>励磁时间 (毫秒)</td>
			<td>速度 (Da/秒)</td>
			<td>时间 (s)</td>
			<td>分辨率</td>
		</tr>
<tr>
<td>Mrm</td>
			<td>积极</td>
			<td>[M + H]+
</td>
			<td>184</td>
			<td>1</td>
			<td>10</td>
			<td>35</td>
			<td>12</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>5.364</td>
			<td>单位</td>
		</tr>
<tr>
<td>MS3
</td>
			<td>负</td>
			<td>[M + HCOO]-
</td>
			<td>M-15</td>
			<td>-26</td>
			<td>-10</td>
			<td>-40</td>
			<td>-</td>
			<td>0</td>
			<td>25</td>
			<td>10000</td>
			<td>自动 caluculated</td>
			<td>单位</td>
		</tr>
</tbody></table>表2。分别用于定性和定量分析的三重四极和四极线性离子阱质谱中 MS3和 MRM 模式的参数.此表是从哈马et改编而来的。6
<img alt="Table 3" src="/files/ftp_upload/57293/57293table3.jpg"> 表3。得到的校准曲线的参数和验证结果.结果由哈马et进行了修改。6
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>SM 的分子种类</td>
			<td>量 (pmol/毫克蛋白)</td>
		</tr>
<tr>
<td>d18:1/14:0</td>
			<td>115。5</td>
		</tr>
<tr>
<td>d16:1/16:0</td>
			<td>115。5</td>
		</tr>
<tr>
<td>d17:1/16:0</td>
			<td>239。8</td>
		</tr>
<tr>
<td>d18:2/16:0</td>
			<td>449。9</td>
		</tr>
<tr>
<td>d18:1/16:0</td>
			<td>3355。1</td>
		</tr>
<tr>
<td>d18:0/16:0</td>
			<td>203。8</td>
		</tr>
<tr>
<td>d18:1/17:0</td>
			<td>106。3</td>
		</tr>
<tr>
<td>d18:2/18:0</td>
			<td>26。5</td>
		</tr>
<tr>
<td>d20:0/16:1</td>
			<td>94。9</td>
		</tr>
<tr>
<td>d18:1/18:0</td>
			<td>94。9</td>
		</tr>
<tr>
<td>d18:2/22:0</td>
			<td>102。3</td>
		</tr>
<tr>
<td>d18:1/22:0</td>
			<td>235</td>
		</tr>
<tr>
<td>d18:1/23:1</td>
			<td>83。3</td>
		</tr>
<tr>
<td>d18:1/23:0</td>
			<td>23。9</td>
		</tr>
<tr>
<td>d18:1/24:2</td>
			<td>286。5</td>
		</tr>
<tr>
<td>d18:2/24:1</td>
			<td>286。5</td>
		</tr>
<tr>
<td>d18:1/24:1</td>
			<td>853。2</td>
		</tr>
<tr>
<td>d18:1/24:0</td>
			<td>94。6</td>
		</tr>
<tr>
<td>d18:1/25:1</td>
			<td>12。9</td>
		</tr>
</tbody></table>表4。HeLa 细胞中每个 SM 物种的数量.HeLa 细胞生长在含有10% 血清的培养基培养基中。细胞的收获, 并提取总脂分数的布莱 & 戴尔方法。结果由哈马et进行了修改。6

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 ...

quantitative-qualitative-method-for-sphingomyelin-lc-ms-using-two

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.7K 次观看.  Teikyo University. 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。

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

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

科研

生物化学

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

生物工程

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

人类血小板的LC-MS分析作为研究线粒体代谢的平台

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

环境科学

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

鞘糖脂抗原的质谱分析

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

免疫与感染

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 ...

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

用于质谱分析的最佳切割温度化合物中嵌入的人组织制备

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

癌症研究

使用稳定同位素标记筛选脂质结构

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (&lt;1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

作者聚焦：使用稳定同位素标记定量复杂脂质组学样品

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从 头 追踪脂质

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...

用两个稳定 Isotopically 标记鞘磷脂种鞘磷脂的定量和定性方法

摘要

探索更多视频

此视频中的章节

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

脂滴隔离定量质谱分析

人类血小板的LC-MS分析作为研究线粒体代谢的平台

鞘糖脂抗原的质谱分析

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

用于质谱分析的最佳切割温度化合物中嵌入的人组织制备

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质

用两个稳定 Isotopically 标记鞘磷脂种鞘磷脂的定量和定性方法

摘要

探索更多视频

此视频中的章节

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

脂滴隔离定量质谱分析

人类血小板的LC-MS分析作为研究线粒体代谢的平台

鞘糖脂抗原的质谱分析

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

用于质谱分析的最佳切割温度化合物中嵌入的人组织制备

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从 头 追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从 头 追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质