Dr. Rawi Ramautar would like to acknowledge the financial support of the Veni grant scheme of the Netherlands Organization of Scientific Research (NWO Veni 722.013.008).

注意:这里描述了使用鞘CE-MS的代谢谱研究该协议仅用于实验室使用。下面列出的程序是根据最近发表的作品4,5。进一步的实验细节，可以在这些文件中找到。此前使用此协议，请所有相关的材料安全数据表（MSDS）。请使用所有适当的实验室安全程序，包括安全眼镜，白大褂和手套，在开展该协议所列的实验时。 1.试剂溶液和样品的制备<ol><li> 背景电解质的制备（BGE） <ol><li>准备新的BGE溶液（10％（V / V）乙酸，pH值2.2）的每一天。 </li><li>加9.0毫升水到10ml玻璃小瓶中，并在通风橱中加入1.0毫升乙酸的水。调匀使用涡旋该溶液中。 </li></ol></li><li> 代谢STANDAR的制备ð混合<ol><li>溶解50微升含有60阳离子代谢成50微升水中的50μM的阳离子标准混合物中，将溶液充分混合。在-80°C储存在不使用时。 </li><li>溶解50微升含有30阴离子代谢成50微升水中的50μM的阴离子标准混合物中，将溶液充分混合。存储此溶液，以等分试样，以防止相同的标准混合物的冷冻/解冻循环，在-80℃下，不使用时。 注:当代谢产物在-80℃妥善保存标准混合物连续3个月保持稳定。 </li></ol></li><li> 提取物对胶质母细胞瘤细胞系的制备 <ol><li>洗粘附人的U-87 MG成胶质细胞瘤细胞三次用1ml冰冷的0.9％氯化钠溶液21中 。 </li><li>加入2毫升冰冷的甲醇/水的溶液（8/2，体积/体积），以贴壁细胞并用橡胶放倒细胞刮刀21刮除。 </l我&gt; <li>收集2分钟在管的甲醇/水的溶液和ultrasonicate。 </li><li>在16100 xg离心4℃10分钟加氯仿至甲醇/水级分（最终比8/8/2，体积/体积/体积）并离心样本。 </li><li>收集甲醇/水层，并用真空浓缩器蒸发该馏分。通过鞘CE-MS重组在50μl水中分析干燥的材料。在不使用时存放在-80℃的样品。 </li></ol></li></ol> 2.设置鞘CE-MS系统<ol><li> 裸石英墨盒安装与多孔尖端发射极 <ol><li>将新的裸石英墨盒在CE仪器的多孔尖端发射极（共30微米内径×90厘米长）。 </li><li>在50psi施加向前漂洗用100％甲醇的软件15分钟控制CE仪器并进行目视检查液体是否从capil流出在此冲洗步骤毛细管出口。此外，在相反的方向上在50psi进行漂洗使用BGE溶液可视地检查液体是否从导电毛细管流出5分钟。 </li><li>重复步骤2.1.2在万一没有液滴形成已在毛细管出口被观察到100磅的压力。如果在该步骤中没有观察到液滴形成安装一个新的裸石英盒。 </li><li>在50psi 10分钟用水冲洗分离毛细管在50psi进行10分钟，随后用0.1 M氢氧化钠在50psi进行10分钟，然后用水，最后在50与BGE psi的10分钟。 注意:步骤2.1.1至2.1.4只需要安装新的毛细管墨盒。 </li></ol></li><li> 毛细多孔尖端发射极到ESI-MS耦合 注:以耦合CE毛细管MS，确保MS仪器被校准并连接到CE系统之前。设置ESI电压0适合用纳米喷雾源的MS仪器。气体1，天然气2接口加热器温度在非常低的流量并没有用作ESI只把它运用离子喷雾电压设置为1500 V.设置窗帘在5 PSI发生。 <ol><li>从水管除去石英盒的喷雾器尖端和在纳米喷雾源适配器安装它用于耦合到MS仪器。确保在CE仪器的BGE小瓶的高度匹配喷雾器尖端的高度。 </li><li>通过在50psi与BGE漂洗5分钟检查通过导电毛细管液流。在此冲洗步骤在ESI喷雾器针的基部的液滴形成应观察。 </li><li>冲洗用BGE分离毛细管在50psi在向前方向10分钟。液滴形成应在多孔尖端发射极（喷雾器尖端）的前端在该步骤中被观察到。 </li><li>在C的距离的多孔尖端发射极到MS入口的入口位置IRCA 2〜3毫米。 </li><li>用1分钟的升温时间申请30千伏的电压，并开始在m / z范围获取MS数据从65至1000 米/ z计算使用0第一电喷雾电压代谢谱的研究。 注:质谱应该是无效的信号，因为应该没有电。 </li><li>设置ESI电压为1,000V，而测量数据的携带。加大与200伏增量ESI电压直到恒定背景信号观察至少15分钟。 </li><li>通过在x，y或z方向，以查看哪个位置提供的最大的和最稳定的MS信号（总离子电泳）移动它优化相对于该MS入口中心的多孔尖端发射极的位置。 </li><li>优化多孔尖端发射极的位置和确定最佳ESI电压后，设置ESI电压为0V，并使用了5分钟的升温时间为30千伏降低CE的电压为1千伏。 </li><li>创建一个MS法全光照克最佳ESI电压在CE仪器的代谢物标准和生物样品分析的方法CE。 </li></ol></li></ol> 3.代谢物标准和生物样品分析<ol><li> 在鞘CE-MS系统的性能评价 <ol><li>转让20微升的阴离子代谢物的标准混合物成适合在CE小瓶中，并把这个小瓶在进口样品盘空100微升microvial（PCR瓶）。 注:在microvial一个可靠的注射所需的最小体积为2微升。 <ol><li>启动STEP 2.2.9期间创建的MS采集负离子模式的方法，并随后开始使用软件控制CE仪器的CE序列。 </li><li>在1.0磅10冲洗BGE分离毛细管在50psi 3分钟，然后注入在2.0磅60秒（对应于毛细管体积的3％20 NL），然后通过BGE注射秒。 注意:此后，MS数据采集被触发。 </li><li>适用-30千伏的电压为1.0分钟，0.5磅的用于在入口30分钟的压力的斜坡时间。 30分钟电泳分离后，停止MS数据采集和利用5分钟的升温时间（CE电压的逐渐减少电泳分离提高了多孔尖毛细管发射器的耐用性之后）降低CE电压为-1千伏。 </li></ol></li><li>样品进样之间，用清水冲洗毛细管，0.1M氢氧化钠，水和BGE各在30psi 3分钟。 </li><li>通过确定迁移时间和所分析的阴离子代谢物的混合物的信号强度分析所记录的数据。 </li><li>评估阴离子代谢物标准是否10和28分钟之间出现在该地区。 </li><li>检查是否三种结构相关的异构体， 即 ，D-葡萄糖-1-磷酸，D-葡萄糖-6-磷酸和D-果糖-6-磷酸是部分分离， 即 ，前两个峰之间的分辨率为大约0.75和最后两个峰的是大约0.50（ 见图1）。 注:1.5的决议表明了两个相邻峰的基线分离。 </li><li>重复步骤3.1.1和3.1.2阳离子代谢的混合物。确保MS检测现在处于正离子模式和CE电压为+30千伏。 </li><li>通过确定迁移时间和所分析的阳离子代谢物的混合物的信号强度分析所记录的数据。 </li><li>评估阳离子代谢物标准是否出现在8和22分钟之间的区域。检查异亮氨酸和亮氨酸是否迁移15和15.5分钟之间，并确定如果分辨率大约0.5。 </li></ol></li><li> 生物样品的分析 <ol><li>重复步骤3.1.1和3.1.2的胶质母细胞瘤细胞系的提取物的阴离子代谢轮廓。 注:代谢CON样品预处理之后获得10吨对应于大约20个细胞/ NL的细胞密度，因此，一个20 NL注射是每次分析400个细胞的等价物。 。 </li><li>创建用于使用5 MDA质量精度的代谢产物乳酸（M / Z 89.0243）提取离子电泳，并检查该信号强度是否高于100,000计数。 </li><li>重复步骤3.1.1和3.1.2的胶质母细胞瘤细胞系的提取物的阳离子代谢轮廓。 注:一个高度信息丰富的总离子电泳应该在此模式下20 NL注射液被观察到。 </li><li>的分析或在不使用时后，用水冲洗所述毛细管在50psi进行15分钟，并在毛细管的入口部分存储在一个含有小瓶水和多孔质部（出口部）中的管也含有水。 </li></ol></li></ol>

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We have nothing to disclose.

采用多孔尖端发光体A鞘CE-MS法已经提出了高极性和电荷的代谢物的分析。这种方法的独特之处在于阴离子或阳离子代谢产物只能由切换MS检测和CE电压极性进行概要。广泛的生物样品中的高极性和电荷的代谢物能够以高的分离效率，这是结构上相似的代谢物是至关重要的，并与在（低）纳摩尔范围检测限进行分析。所提出的协议集中在使用鞘CE-MS的对于细胞提取物的代谢谱，以举例说明用于生物样品的代谢谱的方法的效用。此处所描述的方法也可以用于其它类型的生物样品的代谢谱，如人尿5，考虑到一个适当的样品预处理过程被使用。 该鞘CE-MS方法具d被基于一个多孔尖端发射极，它允许CE的固有的低流动性的用法。在这种情况下，稳定的ESI信号是一个先决条件可再现代谢谱的研究。因此，重要的是，喷雾器尖端被正确地定位在MS入口的前面。在此设置中，在ESI过程主要依赖于BGE的性质，因此，BGE优化是非常关键的。所述鞘结构相比，可加入各种鞘液体组合物以提高离子化效率鞘液的CE-MS系统少的通用性。所述鞘ESI喷雾器针需要完全填充导电液体（ 即 ，BGE溶液）。不稳定ESI信号可能导致从部分或完全堵塞毛细管。在与BGE高压冲洗就可以解决这个问题。否则分离毛细管需要更换。在此之前的分析性能考核，稳定的ESI背景信号应产生的第一是从一天到另一相一致。 需要使用标准的代谢产物为混合物，每天检查鞘CE-MS方法进行代谢谱研究的分析性能。在相同的实验条件下，一致的迁移时间， 即低于3％的变化对于内天（N = 10）和日间（N = 5），使用的代谢物标准混合物（12.5μM）的20 NL注射，峰高度/地区（低于15％的变化）和车牌号码（60,000至400,000范围）应获得。检测限应该在纳摩尔范围对大多数代谢物标准。只有当这些条件都满足是准备用于生物样品的代谢谱的方法。如果不是，则MS仪器需要调整和重新校准或需要更改多孔尖端毛细管发射极。 CE-MS之间的一种有效的漂洗步骤的分析是非常重要，不仅是为了防止潜在的交叉污染也能保持分离性能。潜在交叉污染可通过污染与样品，因此通过置换新BGE小瓶解决BGE小瓶引起。当鞘CE-MS法是不使用时，它断开分离毛细管并存储在水中的毛细管，并与在含有水以延长毛细管寿命的管保护套筒浸没外侧的入口侧是重要的。 总之，所提出的无鞘CE-MS法示出了根据在此协议报告的过程中使用时，生物样品的代谢谱的巨大潜力。在这个阶段，实验室间比数据肯定，以评估这种做法对代谢的（长期）的重现性和稳健性需要鞘CE-MS。该协议可能会刺激这样的研究。各种分析挑战仍需要考虑。为了获得最佳的PERFormance，在CE电流应该优选低于5μA和在此阶段仅在91厘米的长度，有可能妨碍高通量测定法的发展提供的毛细多孔尖端发射器被保持。另外，用于阴离子代谢轮廓这可能不是最优化的用于实现结构相关的糖磷酸酯的基线分离低pH分离缓冲液。同样重要的是，只有阴离子代谢产物可以分析这些（部分）带负用过的分离条件下充电。下一步骤是为目前评估鞘CE-MS法用于临床代谢谱研究的效用，单一多孔尖端的毛细管发射器只能用于多达100生物样品的分析。 总体来看，在鞘CE-MS方法的进一步发展将在代谢组学， 即领域打开一个新的方向，朝中的生物功能有更深的了解充裕的限制情况。

在当代代谢，高端分析分离技术被用来进行分析，以获得一个代表广泛代谢物类的读出的生物体1的生理状态。代谢组学研究的最终目标是获得一个答案，一个给定的生物/临床问题。目前，人类代谢组数据库包括代表内源性和外源性化合物（从营养物，微生物群，药物和其它来源后者始发）4万多代谢物的条目2。定在物理 - 化学性质和这些代谢物的浓度范围的巨大多样性，不同的分离机制的多个分析技术应结合以轮廓尽可能多的代谢物尽可能给定生物样品中的使用。例如，Psychogios 等人使用五个分析分离技术的组合为代谢教授导致检测4000多个不同的化学代谢物3的人血清iling。 在本文中，将注意力投放到最近开发的CE-MS策略生物样品4,5的代谢谱。在CE中，更具体地毛细管区带电泳（CZE;通常称为CE）化合物是分离的它们的电荷多尺寸比率的基础上，因此，此分析技术是非常适合于极性及带电代谢物的分析。 CE的分离机理是从基于色谱的技术根本不同的，由此提供对生物样品6-8的代谢组合物的互补视图。曾我和同事是第一个显示的CE-MS的效用为代谢物的生物样品9,10在全局分析。到现在为止，CE-MS的代谢组的可行性和效用已得到广泛证实11-15。CE经由鞘液接口技术16,17通常耦合到MS;然而，由于由鞘液的毛细管流出的稀释，检测灵敏度固有损害。 最近，证明的是，使用一个无鞘接口相比，CE-MS利用经典鞘-液界面5,18,19显著改善本多种生物样品中的代谢物的检测范围。例如，被人体尿液中鞘CE-MS检测到大约900分子特征，而与鞘液CE-MS 5中观察到约300分子特征。所使用的无鞘接口是基于多孔尖端发射极，其通过Moini 20发明，使得在组合的有效使用的CE的固有的低流动性的纳米ESI-MS。 为了刺激代谢组学领域的使用鞘CE-MS的，提出的协议描述该方法如何可用于生物样品中的高极性代谢物的分析，作为例示用于从成胶质细胞瘤细胞系提取物的分析。它表明，也可用于使用完全相同的毛细管和分离条​​件，从而减少分析时间和的全局分析提供一个单一的分析平台的阴离子代谢物的分析阳离子代谢物的分析的无鞘CE-MS法充电代谢物。该协议还介绍了与MS仪器鞘多孔尖端发射极的有效调整的策略。

<table><tbody><tr><td>CESI 8000 instrument</td><td>Sciex</td><td>A98089</td><td>OptiMS adapter required to couple CESI to MS</td></tr><tr><td>OptiMS Fused-Silica Cartridge, 30&amp;nbsp;&amp;mu;m ID x 90&amp;nbsp;cm total length</td><td>Sciex</td><td>B07367</td><td></td></tr><tr><td>OptiMS Adapter for Sciex Nanospray III source</td><td>Sciex</td><td>B07363</td><td></td></tr><tr><td>CESI vials</td><td>Sciex</td><td>B11648</td><td></td></tr><tr><td>Micro vials</td><td>Sciex</td><td>144709</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma</td><td>A6283</td><td>Use in fume hood</td></tr><tr><td>Cationic metabolite mixture</td><td>Human Metabolome Technologies</td><td>H3304-3034</td><td></td></tr><tr><td>Anionic metabolite mixture</td><td>Human Metabolome Technologies</td><td>H3304-1031</td><td></td></tr><tr><td>Methanol (LC-MS Ultra Chromasolv)</td><td>Sigma</td><td>14262</td><td>Use in fume hood</td></tr><tr><td>Sodium hydroxide solution</td><td>Sigma</td><td>72079</td><td>0.1 M</td></tr><tr><td>U-87 MG Glioblastoma cell line</td><td>Sigma</td><td>89081402</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>650498</td><td>Toxic; use in fume hood</td></tr></tbody></table>

sheathless capillary electrophoresis&#8211;mass spectrometry for metabolic profiling of biological samples

在代谢组学，广泛的分析技术被用于复杂样品中（内生）的代谢产物全球分析。在本文中，一个协议被提出用于通过毛细管电泳 - 质谱（CE-MS）的生物样品中的阴离子和阳离子的代谢物的分析。 CE是非常适合高极性带电代谢物的分析化合物的电荷 - 大小比率的基础上分离。最近开发的鞘接口设计， 即，多孔尖接口，用于连接CE电喷雾（ESI）MS。这种接口方法允许组合的有效使用的CE的固有的低流动性的与MS，导致对大范围的极性代谢物类纳摩尔检测限。这里介绍的协议是基于在低pH的分离条件采用裸熔融石英毛细管与多孔尖端发射极对的一个分析生物样品中的代谢物类宽阵列。它证明了相同的无鞘CE-MS法可以仅切换MS检测和可用于阳离子代谢物，包括氨基酸，核苷和小肽，或阴离子的代谢产物，包括糖磷酸盐，核苷酸和有机酸，的分析分离电压极性。各种生物样品中的高度信息丰富的代谢概况，如尿，脑脊髓液和胶质母细胞瘤细胞系的提取物，可通过该协议中的CE-MS分析的不到1小时来获得。

所提出的无鞘CE-MS法是能够提供高效率的，即，塔板数范围从60,000至400,000，在用10％乙酸（pH为2.2）作为BGE纳摩尔检测限阴离子和阳离子的代谢物分布。对于高极性阴离子代谢物的分析方法的分离性能是展示三个结构相关的糖磷酸异构体（ 图1）。虽然这三个分析物不能获得基线分离，部分分离足以允许其被MS选择性检测这些分析物具有相同的精确质量。所述鞘CE-MS法的对数细胞的限制，代谢谱的电位即 20 NL喷射对应于400个细胞（细胞密度为大约20个细胞/ NL）中，展示了阳离子代谢物中的提取物的分析在胶质母细胞瘤细胞系（图2），其中上述一个的S / N比≥5检测到超过300个分子的功能。 <img alt="图1" src="/files/ftp_upload/54535/54535fig1.jpg" /> 图1.糖磷酸异构体的分析鞘CE-MS提取离子为负离子模式与鞘CE-MS得到的三种糖磷酸异构体（25μM）电泳。实验条件:BGE，10％乙酸（pH为2.2）;分离电压，-30千伏（+0.5磅施加在CE毛细管入口）;样品注射，2.0磅60秒。经许可后转载4。 <a href="https://www.jove.com/files/ftp_upload/54535/54535fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54535/54535fig2.jpg" /> 图2.异亮氨酸和L的分析通过鞘CE-MS提取离子的正离子模式与鞘CE-MS获得的两个氨基酸的异构体（25μM）电泳eucine。实验条件:BGE，10％乙酸（pH为2.2）;分离电压，+ 30千伏;进样，2.0磅，持续60秒。 <a href="https://www.jove.com/files/ftp_upload/54535/54535fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54535/54535fig3.jpg" /> 图3. 鞘CE-MS的潜力在细胞系提取物分析阳离子的代谢物。代谢轮廓（总离子电泳）在正离子模式下与鞘CE-MS成胶质细胞瘤细胞系的提取物观察到的。实验条件:BGE，10％乙酸（pH为2.2）;分离电压，+ 30千伏;样品注射，2.0磅60秒。经许可后转载4 </s了&gt;。 <a href="https://www.jove.com/files/ftp_upload/54535/54535fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Sheathless Capillary Electrophoresisâ€“Mass Spectrometry for Metabolic Profiling of Biological Samples at JoVE.com

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

使用鞘多孔提示界面设计的毛细管电泳 - 质谱联用生物样品的代谢谱的协议提出。

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

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Biochemistry

11.6K 次观看.  Leiden University. 使用鞘多孔提示界面设计的毛细管电泳 - 质谱联用生物样品的代谢谱的协议提出。 

视频: Sheathless Capillary Electrophoresis–Mass Spectrometry for Metabolic Profiling of Biological Samples

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

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

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

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

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

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

科研

化学

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

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

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

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

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

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

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

生物化学

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

毛细管电泳质谱法提取培养细胞中的水代谢物进行代谢组学分析

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

癌症研究

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

活蛙 (爪蟾) 胚胎单细胞代谢的微探针毛细管电泳质谱

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

纳米材料获得的蛋白质和代谢物科罗纳的毛细电电量谱方法

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...