作者感谢 Cesar Ramirez 博士在初始方法开发过程中的贡献。这项研究由捷克共和国捷克科学基金会向明尼苏达州提供的 NIAID 赠款 22-21244S 项目 R21AI167849 资助。

1. 样品制备
<ol><li>昆虫解剖<ol><li>使用微针解剖 埃及伊蚊 卵巢，当它们 7 天大时，在磷酸盐缓冲盐水溶液中。</li>
		<li>使用微针从磷酸盐缓冲盐水溶液中收集八个卵巢，并储存在 -80 °C 的 1.5 mL 微量离心管中。</li>
		<li>在生物重复中收集卵巢。</li>
	</ol></li>
	<li>脂质提取<ol><li>将 10 μL 内标 （ISTD） 加入收集的 8 个卵巢中，浓度为 1 ppm。ISTD 是一种标记的脂质混合物，含有 7 个 1-十五烷酰-2-油酰-sn-甘油-3-磷酸胆碱-D7 的氘（15： 0-18：1 （D7） PC）、溶血磷脂酰胆碱-D7 （18：1 （D7） Lyso PC）、1-十五烷酰-2-十八碳烯酰-甘油-3-磷酸乙醇胺-D7 （15：0-18：1 （D7） PE）、1-油酰-2-羟基-sn-甘油-3-磷酸乙醇胺-D7 （18：1 （D7） Lyso PE）、1-十五烷酰-2-油酰-sn-甘油-3-磷酸肌醇-D7 （15：0-18：1 （D7） PI）、1-十五烷酰-2-油酰-sn-甘油-3-磷酸-L-丝氨酸-D7 （15：0-18：1 （D7） PS）、1,2-二十五碳酰基-3-十八碳烯酰-sn-甘油（15：0-18：1-15：0 （D7） TAG）、1-十五碳酰-2-十八碳烯酰基）-sn-甘油-D7 （15：0-18：1 （D7） DAG）、2-十八碳烯酰-sn-甘油-D7 （18：1 MAG （D7））、胆固醇油酸酯-D7 （18：1 （D7） 胆固醇酯）。</li>
		<li>等到室温干燥。加入 100 μL 丁醇/甲醇和 3 μL 丁基化羟基甲苯。用 1.5 mL 聚丙烯研杵手动均质 10 秒。</li>
		<li>用 200 μL 丁醇/甲醇冲洗研杵，并与均质溶液混合。</li>
		<li>在室温和中等功率下对微量离心管进行超声处理 30 分钟。</li>
		<li>在 20 °C 下以 1600 x g 离心管 10 分钟。 将上清液转移至装有 300 μL 硅烷化玻璃内插管的自动进样器样品瓶中。</li>
	</ol></li>
</ol>2. 仪器设置
<ol><li>制备流动相<ol><li>使用乙腈、水和异丙醇作为相应比例制备成分为 30：40：30（流动相 A）和 10：5：85（流动相 B）的流动相。在两种流动相中，加入 10 mmol/L 甲酸铵的水溶液和 0.1% 甲酸，以形成缓冲条件。</li>
		<li>称取 3.15 g 甲酸铵溶于 10 mL 水中。在 12.5 mL 水中加入 500 μL 此溶液。依次放入 250 mL 容量瓶中，用异丙醇填充至培养瓶的约 2/3 处。旋转混合。</li>
		<li>加入 250 μL 甲酸 （85%），加入 25 mL 乙腈和异丙醇至 250 mL 填充线，然后混合。</li>
	</ol></li>
	<li>在 MS 系统中，在屏幕左侧的 MS 设置中，通过选择 50 作为最小值，选择 1850 作为最大扫描设置来确定质量范围。<ol><li>选择 Polarity （极性） 作为正离子模式。选择扫描模式作为 DIA-PASEF。为此，请选择 Tune and open analysis method from a previous DDA method （ 优化 并使用以下 LC 参数从以前的 DDA 方法中打开分析方法）。<ol><li>对于梯度分离： 样品进样：0% B，保持时间 1 min。 增加至 55% B，保持至 6.4 分钟。 增加至 65% B，保持至 24 分钟。 增加至 88% B，保持至 40.8 分钟。 增加至 95% B，保持至 48.1 分钟。 维生素 B 降至 35%，保持至 56 分钟。 B降至 0%，保持至 60 分钟。</li>
			<li>对于 HPLC 条件： 进样体积：5 μL 溶剂速率：0.25 mL/min 总运行时间：60 min，其中 5 min 用于预处理色谱柱。</li>
			<li>通过将色谱柱插入液相色谱仪来制备液相色谱仪。运行空白以清洁色谱柱并测试是否有渗漏。这可以从仪器不保持压力或流量中看出。</li>
		</ol></li>
	</ol></li>
	<li>要创建新方法，请执行以下步骤 （图 1 和 图 2).<ol><li>打开软件并单击 Connect all instruments（连接所有仪器）。如果连接成功，液相色谱系统将发出一声蜂鸣声，控制屏幕在“状态”框中列出 HyStar，没有任何错误消息。</li>
		<li>单击 Acquisition 选项卡。在 Sample Table Navigator 中，找到 tag 下拉菜单并选择以前的方法。</li>
		<li>将最新样品表加倍。单击 Save As...（另存为... ）并保存新样品列表，格式为：YYYYMMDD_LIP_C30_（XXXX），其中 （XXXX） 是分析的简短描述符。</li>
		<li>右键单击分色方法，然后单击 Edit Method （编辑方法）。单击 Edit Module Method。</li>
		<li>点击 Download。如果成功，将弹出 Download succeeded。关闭屏幕而不保存方法。</li>
		<li>在 MS 设置下，转到 TIMS 设置，选择 1/k0 从 0.70 开始，1/k0 结束于 1.85。将斜坡时间设置为 150 毫秒。这样，占空比和斜坡速率就会自动调整。</li>
		<li>在底部窗口中，选择 Source 选项卡。选择源类型作为 ESI ，并将端板偏移设置为 800 V，将毛细管设置为 4500 V。检查雾化器盒并将压力设置为 4.0 bar。将干燥气体设置为 4.0 L/min，将干燥温度设置为 250 °C。</li>
		<li>在 source 选项卡的右侧，选择 Tune 选项卡。单击 General Sub 选项卡，然后在传输标题下确保以下参数： 偏转 1 delta：70 V 漏斗 1 RF：250 vpp isCID 能量：0 eV 漏斗 2 RF：250 vpp 多极射频：500 vpp。<ol><li>在 collision cell （碰撞单元） 标题下，确保以下参数： 碰撞能量：6.0 eV 碰撞 RF：1200 vpp。</li>
			<li>在四极杆标题下，确保以下参数： 离子能量：6 eV 低质量：250 m/z。</li>
			<li>在 focus pre-TOF 选项卡下，确保以下参数： 传输时间：45 μs 预脉冲存储：5.0 μs。</li>
		</ol></li>
		<li>单击 Processing Sub 选项卡。在质谱峰检测下，请确保以下参数： 绝对阈值：667 绝对阈值（每 100 毫秒 AccuTime）：445。<ol><li>在迁移率峰值检测下，请确保以下参数： 绝对阈值：30.00。</li>
		</ol></li>
		<li>单击 TIMS 子选项卡。在 offsets 选项卡下，确保以下参数： Δt1：-20 伏 Δt2： -150 伏 Δt3：70 伏 Δt4：150 伏 Δt5：0 伏 Δt6：150 伏 碰撞池：300 V。</li>
		<li>在 tune 选项卡的右侧，单击 MS/MS 选项卡。在母离子选项卡下，确保以下参数： PASEF 坡道数量：8 最低收费：1 最大充电量：1.<ol><li>在 Schedule 选项卡下，单击 Advanced 并确保以下参数： 强度：0,1500、5000、10000 重复：1、10、5、1 强度阈值：1500。</li>
			<li>在 active exclusion （活动排除） 选项卡下，确保以下参数： 主动排除复选标记 释放时间：0.4 分钟</li>
			<li>在 collision energy settings （碰撞能量设置） 下，确保以下参数： K0 （V-s/cm3）：0.70、1.60 碰撞能量 （eV）：20.00， 35.00。</li>
			<li>在隔离宽度设置下，确保以下参数： 质量（米/z）：622， 1222 宽度 （m/z）：1.00 2.50。</li>
		</ol></li>
		<li>DIA -PASEF 设置：从左侧的 MS 设置扫描模式中选择 DIA-PASEF 。在 MS/MS 选项卡下，选择 Window Editor。在这里，确保以下参数（图 3）： 窗口设置： 质量宽度：50 Da;质量重叠：0.0 Da;Calculate from polygon： mass steps;移动窗口数：1。</li>
	</ol></li>
</ol>3. 校准
<ol><li>仪器校准（图 4）<ol><li>在 Calibration 子选项卡下，单击 m/z，从下拉框中选择 Tuning Mix ，然后单击右下角的 Calibrate 。</li>
		<li>继续单击 Calibrate（校准）， 直到达到 100% 的分数。</li>
		<li>现在选择 Mobility 选项卡并按照与上述相同的步骤操作，直到达到 100% 校准分数。</li>
	</ol></li>
	<li>校准曲线<ol><li>加标脂质体内标混合物，含 10 μL 氘代内标混合物。 使用 0.1-500 ng/mL 标准混合物和 10 μL 氘代内标混合物的 7 个校准点。</li>
		<li>根据2 中已经描述的 PASEF MS/MS 模式手动注释脂肪酸链。</li>
	</ol></li>
	<li>SIL 贴标效率<ol><li>计算含 SIL 同位素的面积与不含 SIL 的同位素的面积之比。</li>
		<li>通过确定在任何位点找到标记原子的概率来计算理论模式。</li>
		<li>通过将实验同位素谱与数据分析软件上模拟的理论模式拟合来计算 SIL 富集。</li>
	</ol></li>
	<li>开始运行<ol><li>单击 Thermometer 按钮激活柱温箱。通过单击 Valve 按钮激活泵。</li>
		<li>将 MS method （MS 方法） 设置为之前保存的方法。确认进样体积为 5 μL，并且第 1 行以 20：1 样品瓶为目标。</li>
		<li>确保样品瓶 20：1 的 IPA/ACN 含量为 50%。保存样品列表。</li>
		<li>启动序列并验证是否成功注射（由系统上显示的消息确认已注入）。</li>
		<li>复制第 1 行并粘贴在下面以生成新行。根据当前实验，用样品填充第 2 行。确保在自动进样器中使用正确的位置。</li>
		<li>如果流动相是新鲜的，则首先通过注入制造商提供的脂质混合物标准品进行峰形测试。与之前的结果进行比较。2 标准品是 1-十五烷酰-2-油酰-sn-甘油-3-磷酸胆碱（15：0-18：1 PC）、溶血磷脂酰胆碱（18：1 溶血酰 PC）、1-十五烷酰-2-十八碳烯酰-甘油-3-磷酸乙醇胺（15：0-18：1 PE）、1-油酰-2-羟基-sn-甘油-3-磷酸乙醇胺（18：1 Lyso PE）、1-十五烷酰-2-油酰-sn-甘油-3-磷酸肌醇（15：0-18：1 PI）、1-十五烷酰-2-油酰-sn-甘油-3-磷酸-L-丝氨酸（15：0-18：1 PS）、1,2-二十五碳酰基-3-十八碳烯酰-sn-甘油（15：0-18：1-15：0 TAG）、1-十五碳酰基-2-十八碳烯酰基）-sn-甘油（15：0-18：1 DAG）、2-十八碳烯酰-sn-甘油（18：1 MAG）、胆固醇油酸酯（18：1 胆固醇酯）。</li>
		<li>确保新行具有正确的分离方法，而不是行 1 中使用的预处理方法。确保 MS 方法正确（当天的方法）。</li>
		<li>检查是否加载了正确的处理方法，以及是否启用了 Run Automated Process 。</li>
		<li>保存样品列表以确保在当前运行完成后分析样品。</li>
	</ol></li>
</ol>4. 数据分析
<ol><li>打开 数据分析 软件（图 5）。在左上角，单击 文件 按钮并选择 打开...</li>
	<li>选择所需的文件。右键单击文件名并选择 Properties （属性）。<ol><li>选择 Calibration Status （图 6）。在下拉框中，为质谱仪选择 Initial Calibration （初始校准 ）。确保所有误差均小于 1 ppm。</li>
		<li>接下来，选择 Initial Mobility Calibration 并确认误差小于 1 ppm。</li>
		<li>右键单击 Chromatogram（色谱图 ），然后选择 Edit Chromatogram（编辑色谱图 ）（图 6C）。在 “类型”中，单击下拉框并选择 “提取离子色谱图 ”（图 6D）。在过滤器下，选择 所有 MS ，在扫描模式下选择 全部。这是为了查看目标分子的峰，而不仅仅是其片段。<ol><li>在此之下，有两个用于分子选择的过滤器选项：Masses 或 Formula。如果选择了 质量 ，则可以选择感兴趣分子的理论 m/z。对于这种情况，请选择 829.7985 m/z。如果选择了 Formula（公式 ），则可以选择分子的公式以及色谱图的目标离子形式。在这种情况下，选择铵 [M+NH4]。这是因为流动相中的铵缓冲液有利于这种离子形式的电离（图 7）。</li>
			<li>对于 width （宽度），选择 ±1。对于极性，请确保它保持正模式。在 Mobility （移动度） 下，选择 1.455-1.465。 这样做是为了过滤掉任何不在这些值范围内的分子并分离出目标分子。</li>
		</ol></li>
	</ol></li>
	<li>选择参数后，单击右上角的 Add > OK 。片刻之后，软件将处理选择并输出色谱图。</li>
	<li>右键单击目标峰起点处的基线，然后按住峰的末尾进行拖动。这种手动积分将提供该保留时间内片段的实时质谱图。</li>
	<li>动能图<ol><li>右键单击 Mobilogram 的屏幕左侧，然后选择 Edit Mobilogram （图 8）。将出现一个名为 Edit Mobilogram Traces 的窗口。遵循相同的步骤 C.e.i.1 到 C.e.i.5（图 9）。</li>
		<li>在 Retention Time （保留时间 ） 处，输入目标峰的保留时间。在本例中为 37.45-37.55 分钟。</li>
		<li>选择参数后，单击右上角的 Add > OK 。片刻之后，软件将处理选择并输出色谱图。</li>
	</ol></li>
	<li>对于提取的离子，重复步骤 4.2.3.1-4.3.2.2，直到形成离子列表。</li>
	<li>在 MS 窗口底部，选择 Profile MS （分析 MS ） 和 Fragment MS（片段 MS）。这将允许获得来自全扫描和 PASEF 的离子的光谱视图。<ol><li>在谱图视图中，右键单击并选择 Copy Compound Spectra（复制化合物谱图）。通过右侧显示的光谱数据，您可以找到有关化合物的更多信息，例如分辨率、强度和信噪比（S/N; 图 10）。</li>
	</ol></li>
	<li>加工<ol><li>要处理数据，请手动积分色谱图和淌度峰，以产生有关目标分子的宝贵信息（图 10）。</li>
		<li>右键单击 Find（查找 ），然后选择 Integrate Only chromatogram （仅整合色谱图） 或 mobilogram（移动图）（图 11）。左键单击并突出显示所需的峰。此信息包括保留时间、面积、信噪比和迁移率。</li>
	</ol></li>
</ol>

使用稳定同位素标记筛选脂质结构

<ol>
	<li>Stokvis, E., Rosing, H., Beijnen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotopically+labeled+internal+standards+in+quantitative+bioanalysis+using+liquid+chromatography/mass+spectrometry:+Necessity+or+not.">Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: Necessity or not.</a> Rapid Commun Mass Spectrom. 19 (3), 401-407 (2005).</li><li>Tose, L. V., 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=Coupling+stable+isotope+labeling+and+liquid+chromatography-trapped+ion+mobility+spectrometry-time-of-flight-tandem+mass+spectrometry+for+de+novo+mosquito+ovarian+lipid+studies.">Coupling stable isotope labeling and liquid chromatography-trapped ion mobility spectrometry-time-of-flight-tandem mass spectrometry for de novo mosquito ovarian lipid studies.</a> Anal Chem. 94 (16), 6139-6145 (2022).</li><li>Meier, F., Park, M. A., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapped+ion+mobility+spectrometry+and+parallel+accumulation-serial+fragmentation+in+proteomics.">Trapped ion mobility spectrometry and parallel accumulation-serial fragmentation in proteomics.</a> Mol Cell Proteomics. 20, 100138 (2021).</li><li>Basit, A., Pontis, S., Piomelli, D., Armirotti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+mobility+mass+spectrometry+enhances+low-abundance+species+detection+in+untargeted+lipidomics.">Ion mobility mass spectrometry enhances low-abundance species detection in untargeted lipidomics.</a> Metabolomics. 12 (3), 50 (2016).</li><li>Meier, F., 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=Online+parallel+accumulation-serial+fragmentation+(PASEF)+with+a+novel+trapped+ion+mobility+mass+spectrometer.">Online parallel accumulation-serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer.</a> Mol Cell Proteomics. 17 (12), 2534-2545 (2018).</li><li>Koopmans, F., Ho, J. T. C., Smit, A. B., Li, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analyses+of+data+independent+acquisition+mass+spectrometric+approaches:+DIA,+WiSIM-DIA,+and+untargeted+DIA.">Comparative analyses of data independent acquisition mass spectrometric approaches: DIA, WiSIM-DIA, and untargeted DIA.</a> Proteomics. 18 (1), 1700304 (2018).</li><li>Fern&#225;ndez-Costa, 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=Impact+of+the+identification+strategy+on+the+reproducibility+of+the+DDA+and+DIA+results.">Impact of the identification strategy on the reproducibility of the DDA and DIA results.</a> J Proteome Res. 19 (8), 3153-3161 (2020).</li><li>Prakash, A., 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=Hybrid+data+acquisition+and+processing+strategies+with+increased+throughput+and+selectivity:+PSMART+analysis+for+global+qualitative+and+quantitative+analysis.">Hybrid data acquisition and processing strategies with increased throughput and selectivity: PSMART analysis for global qualitative and quantitative analysis.</a> J Proteome Res. 13 (12), 5415-5430 (2014).</li><li>Tsou, C. 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=DIA-umpire:+Comprehensive+computational+framework+for+data-independent+acquisition+proteomics.">DIA-umpire: Comprehensive computational framework for data-independent acquisition proteomics.</a> Nat Methods. 12 (3), 258-264 (2015).</li><li>Triebl, A., Wenk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+considerations+of+stable+isotope+labelling+in+lipidomics.">Analytical considerations of stable isotope labelling in lipidomics.</a> Biomolecules. 8 (4), 151 (2018).</li></ol>

Matthew Willetts 和 Melvin A. Park 是 timsTOF 商用仪器制造商 Bruker Daltonics Inc 的员工。作者声明没有竞争性的经济利益。

在评估该方案的使用情况时，必须确保 DIA-PASEF 和 MS/MS 参数适用于所讨论的甘油三酯。具体来说，活动窗口和窗口宽度应遵循甘油三酯的模式。这可以通过使用 DDA 方法的上一次运行来确定。在追踪内标中的甘油三酯时，DIA 设置的窗口应覆盖所有预先确定的目标分子。窗口的大小应相等，范围在所需化合物的 m/z 和 1/k0 范围内（图 7）。如果窗口大小不正确，将跳过要碎裂的...

在分析脂质数据时，稳定同位素标记 （SIL） 是评估生物体代谢途径的有效方法。在这种方法中，分析物中的原子被含有 13C 或 2H 的稳定同位素取代。这些同位素进一步掺入前体中，这些前体随后嵌入脂肪酸中，标记它们所在的区域。使用这些同位素，人们能够识别脂质的分布和代谢，因为它们会与未标记的背景形成对比1。常见的质谱平台无法区分来自内源性分子的信号2。SIL 的成功需要使用超高分辨率的分析工具。液相色谱与高分辨率捕获离子淌度质谱-平行累积顺序碎裂-飞行时间串联质谱 （LC-TIMS-PASEF-TOF-MS/MS） 联用，可分离标记和未标记物质2。LC-TIMS-PASEF-TOF-MS/MS 分析的优势在于，可以通过保留时间 （RT） 和淌度过滤 MS 信号，从而减少内源性分子的潜在干扰，以及所有所需物质的定量和分离2。在 TIMS 中，离子首先在气体流动相上保持静止，然后根据其迁移率释放。这提供了高分辨率的移动图，同时在比早期 IMS 仪器更低的电压下运行3.移动量图是质量电荷与漂移时间的关系图，可用于根据化合物的漂移时间和重叠量来区分化合物4。
通过使用额外的 PASEF 技术，在不牺牲灵敏度的情况下大大提高了测序速度5。PASEF 理论涉及离子的积累，然后按照其迁移率的顺序顺序顺序释放。这是通过积累离子与其淌度平行来实现的。以这种方式积累可以防止离子损失。此外，母离子选择与离子的积累和释放同时发生。使用这种方法，PASEF 在每次 TIMS 扫描期间选择多个串联的母离子，而不是不使用 PASEF 的 TIMS 仪器通常每次扫描选择单个母离子。当母离子在每帧约 100 ms 的 TIMS 扫描中以 2 ms 的峰值同时积累和释放时，信号被放大超过一个数量级，因此信噪比增加3。在 TIMS 中增加 PASEF 可提高 MS/MS 的效率。由于 TIMS-PASEF 与 MS/MS 联用，因此可以实现更高效的碎裂。与传统的 MS/MS 检测不同，母离子信号从 TIMS-PASEF 压缩，碎片离子与母离子3 的 TIMS 洗脱时间和离子淌度位置同时检测。
从质谱仪采集数据时，扫描模式中有一些选项。数据依赖性采集 （DDA） 根据母离子的丰度从 MS1 扫描中选择母离子，然后将其碎裂并执行 MS2 扫描。此扫描模式会碎裂尽可能多的 precursors。DDA 方法是有针对性的，结果将取决于离子选择3。然而，DDA-PASEF 在重复之间的重现性方面经常存在问题。虽然 DDA 是重点分析的出色工具，但复杂混合物的数据采集难度更大6。这是因为只能同时监测少量离子3。数据非依赖型采集 （DIA） 是一种方法，其中 m/z 的预选窗口独立于其强度进行碎裂，并且所有窗口都在7 范围内进行扫描。使用这种扫描模式，整个离子云从 IMS 传输到质谱仪3。在蛋白质组学研究中，与 DDA 相比，这导致在更短的时间内定量了更多的蛋白质。此外，与 DDA 相比，DIA 遗漏的 m/z 值更少。因此，这种替代方案在信噪比的数据重现性和比 DDA 更好的定量方面显示出很大的改进。在这项工作中，我们的目标是将 DIA 实施到 Tose 等人 2 报告的方法。我们提高了信号的可重复性和强度，利用 DDA 和 DIA 采集获得了有关生物样品中 SIL 脂质动员、分布和代谢的进一步和更确凿的信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accucore C30 colum</td>
			<td>Thermo Fisher Scientific</td>
			<td>27826-252130</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Compass Data Analysis</td>
			<td>Bruker Daltonics Inc</td>
			<td>2363525870</td>
			<td>Version 5.2</td>
		</tr>
		<tr>
			<td>d-Glucose-13C6</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>389374</td>
			<td></td>
		</tr>
		<tr>
			<td>eppendorf tubes</td>
			<td>Fisher Scientific</td>
			<td>14-282-300</td>
			<td></td>
		</tr>
		<tr>
			<td>EquiSplash Lipidomix</td>
			<td>Avanti Polar Lipids</td>
			<td>330731-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>glass vials</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-417-236</td>
			<td></td>
		</tr>
		<tr>
			<td>heavy water (2H2O)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>1.13366</td>
			<td></td>
		</tr>
		<tr>
			<td>polypropylene pestles</td>
			<td>Fisher Scientific</td>
			<td>BAF199230001</td>
			<td></td>
		</tr>
		<tr>
			<td>Prominence LC-20 CE ultrafast liquid chromatograph</td>
			<td>Shimadzu</td>
			<td>L20234651907</td>
			<td></td>
		</tr>
		<tr>
			<td>silanized glass inserts</td>
			<td>Thermo Fisher Scientific</td>
			<td>03-251-826</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose-13C12</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>605417</td>
			<td></td>
		</tr>
		<tr>
			<td>timsTOF</td>
			<td>Bruker Daltonics Inc</td>
			<td>1.84443E+11</td>
			<td></td>
		</tr>
		<tr>
			<td>Tuning Mix Calibration Standard</td>
			<td>Agilent Technologies</td>
			<td>36</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracing de novo lipids using stable isotope labeling lc-tims-tof ms/ms

脂质高度多样化，脂质结构和组成的微小变化会对关键的生物学功能产生深远的影响。稳定同位素标记 （SIL） 为研究脂质分布、动员和代谢以及 从头 脂质合成提供了多种优势。SIL 技术的成功实施需要去除内源性分子的干扰。在本工作中，我们描述了一种用于从生物样品中筛选 SIL 脂质的高通量分析方案;将显示蚊子卵巢发育过程中脂质 从头 鉴定的示例。使用互补液相色谱捕获离子淌度光谱和质谱允许在单次扫描 （<1 h） 中从单个样品中分离和分配脂质。所描述的方法利用了数据依赖性采集和数据非依赖性采集的最新发展，在迁移陷阱中使用平行积累，然后是连续碎裂和碰撞诱导解离。脂肪酸链水平的 SIL 测量揭示了蚊子卵巢发育过程中脂质动力学的变化。脂质 从头 结构根据其保留时间、迁移率和碎裂模式进行可靠分配。

稳定同位素标记有助于在雌性蚊子卵巢的脂质动力学研究中实现甘油三酯的可视化。在 2D 淌度域中，甘油三酯 48：1 显示在与淌度 1/K0 相关的特定保留时间的区域。甘油三酯的强度可以通过较深的蓝色线来显示。其他斑点代表在卵巢中发现的其他甘油三酯。由于质量和结构的差异，保留时间和迁移率也不同，因此出现在图表的不同区域。在区分样品中的甘油三酯时，这些差异是关键（<stro...

作者聚焦：使用稳定同位素标记定量复杂脂质组学样品

这里介绍的是一种利用其保留时间、迁移率和碎裂通过稳定同位素标记来筛选脂质结构的方法。

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 ...

本研究旨在使用稳定同位素标记在脂质组学样品中实现更特异性的定量。这使我们能够了解脂质面板和从昆虫到人类的高度多样化的样品。目前，该领域在脂质组学研究中 LC、TIMS、Top、MS/MS 的标准化方面存在差距。在这里，我们提供了一种为此目的跟踪生物量遗骸的方法。未来，我们的研究将涉及使用 LC 团队工作人员 MS/MS 对生物过程中存在的表观遗传学和代谢组学进行调查，涉及 DDA 和 DIA 方法。

tracing-de-novo-lipids-using-stable-isotope-labeling-lc-tims-tof-msms

Research

JoVE Journal

Chemistry

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

1.1K 次观看.  Florida International University. 本研究旨在使用稳定同位素标记在脂质组学样品中实现更特异性的定量。这使我们能够了解脂质面板和从昆虫到人类的高度多样化的样品。目前，该领域在脂质组学研究中 LC、TIMS、Top、MS/MS 的标准化方面存在差距。在这里，我们提供了一种为此目的跟踪生物量遗骸的方法。未来，我们的研究将涉及使用 LC 团队工作人员 MS/MS 对生物过程中存在的表观遗传学和代谢组学进?...

视频: 作者聚焦：使用稳定同位素标记定量复杂脂质组学样品

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

定量蛋白质组学使用还原二甲基化的稳定同位素标记

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

科研

化学

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

点击化学可视化植物木动力学: 双标记是幸福!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

LC-MCYMS 诊断碎裂过滤的天然产物发现: 微囊藻毒素分析的应用

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

使用 QuEChERS 方法和 C-MS/MS 分析鳄梨中的农药

Determination of 45 Pesticides in Avocado Varieties by the QuEChERS Method and Gas Chromatography-Tandem Mass Spectrometry

Gas chromatography (GC) tandem mass spectrometry (MS/MS) stands as a preeminent analytical instrument extensively employed for the surveillance of pesticide residues in food. Nevertheless, these methods are vulnerable to matrix effects (MEs), which can potentially affect accurate quantification depending on the specific combination of analyte and matrix. Among the various strategies to mitigate MEs, matrix-matched calibration represents the prevailing approach in pesticide residue applications due to its cost-effectiveness and straightforward implementation. In this study, a total of 45 representative pesticides were analyzed in three different varieties of avocado (i.e., Criollo, Hass, and Lorena) using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate and GC-MS/MS. 
For this purpose, 5 g of the avocado sample was extracted with 10 mL of acetonitrile, and then 2.5 g of ammonium formate was added to induce phase separation. Subsequently, the supernatant underwent a cleanup process via dispersive solid-phase extraction employing 150 mg of anhydrous MgSO4, 50 mg of primary-secondary amine, 50 mg of octadecylsilane, 10 mg of graphitized carbon black, and 60 mg of a zirconium oxide-based sorbent (Z-Sep+). The GC-MS/MS analysis was successfully performed in less than 25 min. Rigorous validation experiments were carried out to assess the performance of the method. The examination of a matrix-matched calibration curve for each variety of avocado revealed that the ME remained relatively consistent and less than 20% (considered as a soft ME) for most pesticide/variety combinations. Furthermore, the method&#180;s limits of quantification were lower than 5 &#181;g/kg for all three varieties. Finally, the recovery values for most pesticides fell within the acceptable range of 70-120%, with relative standard deviation values below 20%.

使用 QuEChERS 方法和甲酸铵进行鳄梨样品制备，用于农药分析

The present protocol describes the analysis of multiclass pesticide residues in avocado varieties using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate, followed by gas chromatography-tandem mass spectrometry.

QuEChERS法-气相色谱-串联质谱法测定牛油果品种中45种农药

Gas chromatography (GC) tandem mass spectrometry (MS/MS) stands as a preeminent analytical instrument extensively employed for the surveillance of ...

组蛋白组学分析和翻译后修饰表征

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

作者聚焦： 通过 LC-TIMS-TOF MS/MS 和 PASEF 增强组蛋白 PTM 异构体鉴定

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

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