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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+family+of+animal+sphingomyelin+synthases.">Identification of a family of animal sphingomyelin synthases.</a> EMBO J. 23 (1), 33-44 (2004).</li><li>Kihara, Y., Mizuno, H., Chun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophospholipid+receptors+in+drug+discovery.">Lysophospholipid receptors in drug discovery.</a> Exp Cell Res. 333 (2), 171-177 (2015).</li><li>Kihara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+degradation+pathways,+functions,+and+pathology+of+ceramides+and+epidermal+acylceramides.">Synthesis and degradation pathways, functions, and pathology of ceramides and epidermal acylceramides.</a> Prog Lipid Res. 63, 50-69 (2016).</li><li>Merrill, A. H., Sullards, M. C., Allegood, J. 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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:737px;" width="738">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</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)。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. ...

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

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

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

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

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

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 ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

可溶性和不溶性杭丁顿蛋白的分离分解

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

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

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

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 ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

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 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

EWS-FLI1凝聚物在DNA上组装的单分子成像

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 ...