这项工作是由美国国立卫生研究院授予RR021905 P20 - 01，CF RPD授予STANTO07R0，以及美国航空航天局授予NNH09ZNE002C。

<img src="/files/ftp_upload/2664/2664fig1.jpg" alt="图1"/> 图1。SESI - MS细菌挥发物的分析原理。到SESI反应室（2）细菌培养的顶空的CO 2（1）流离失所。由于挥发物遍历他们通过电云通SESI反应室，并成为离子（3）。一旦离子，挥发性物质进行分析的质谱仪（4）拉进。多余的载气和未反应的细菌挥发物0.22微米过滤器（5）通过，作为额外的保护措施，排放到一个化学罩。插图：SESI电针是一种石英毛细管用削尖的针尖（40微米ID）。 作为SESI - MS使用示范细菌挥发物， 大肠杆菌的表征大肠杆菌 K12和P.有氧假单胞菌 PAO1培养于50 mL LB -伦诺克斯24小时在37 ° C和顶空挥发物SESI - MS谱是在2分钟内收集。二氧化碳在2 L / min的流速（99.99％）用作载气挥发反应室提供。 SESI反应室的建造和安装一个API - 3000（SCIEX公司），取代了原有的电喷雾离子源是自定义。光谱收集使用0.1％甲酸，5.0％甲醇，和94.9％的水，电的解决方案（V / V）正离子模式，交付5 NL / S通过非导电硅胶毛细管用削尖的针尖（40微米ID）。外加电压为2.5千伏。分析师1.4.2软件（Applied Biosystems公司）是用于数据收集与下列参数：20 - 500大，MCA模式，40扫描，3秒/扫描，总的分析时间和2分钟。 1。培养系统<ol><li>选择适当的船只越来越多的文化，在你的实验（例如，通风，光线，温度等），以及质谱仪挥发性物质的效率考虑物种的生长要求。我们选择使用的培养瓶100毫升标准配备了至少有两个螺口的螺纹盖的耐热媒体瓶。通过一个入口的行插入一个载气运送到样瓶和插座线是通过仪器提供的挥发性有机化合物（图1）的另外一个端口插入露儿端口。任何额外的端口堵塞。 </li><li>培养的样品之前，加压船只和淹没在水中，检查有无泄漏。煤气泄漏是非典型薄弱或不存在的离子信号波动的形式结果的主要原因。 </li></ol> 2。生物实验：设置和安全方面的考虑<ol><li>发展适合你的假设条件的文化。这是建议，至少有两个生物的复制，每个有两个技术复制，用于为每个变量。 </li><li>准备一个空白，为每一种文化条件（培养基，抗生素等），并根据您的样品相同的条件下孵化的空白。 </li><li>聘请适合您使用的是生物制剂，考虑到物种的生物安全水平（S）的安全防范措施。 </li><li>为了防止污染仪器的和可行的生物制剂的气体传输线，安装到载气行适当孔径的过滤器。过滤器会不会干扰的挥发物转移到SESI反应室，但可能会稍微影响气溶胶传输效率6。 </li><li>使用辅助遏制或生物安全柜时，附加气调第您的培养瓶中，在溢出生物制剂的情况下，以保证正确的遏制。 </li><li>载气流量的方式，将瓶内的压力无法建立启动和终止样瓶。 </li></ol> 3。仪器优化注：SESI - MS是专门设计的样品挥发物，所以限制使用仪器前香个人护理用品（例如，古龙水，漱口水，乳液，衣物柔顺剂），口香糖，香烟等的使用。紧紧地盖在实验室中所有挥发性化学物质，并在测试过程中尽可能地控制空气的草稿。 以下的仪器参数，这些都影响信号强度和稳定性，将需要为您的仪器和实验进行了优化。 <ol><li>类分子要定位电的解决方案和流速：选择适当的电解决方案，考虑到操作仪器的极性（积极或消极的离子模式）和目标化合物的分子性质。在这个实验中的电液是5.0％，0.1％甲酸，甲醇，94.9％的水（V / V），从而提高信号强度弱极性分子whilË提供良好的信号稳定性。解决的办法是发表在5 NL / s的流量</li><li> 载气流量：载气流量可能会影响电稳定性和信号强度。这里使用的CO 2（≥99.99％）在2 L / min的流速。 </li><li> 针形状和位置：针尖形状和位置的强烈影响信号强度和稳定性。当安装一个新的针，针的位置，必须进行优化，以创建一个低的背景下，高分析物信号强度和信号稳定之间的平衡。为了重现SESI针更改后的光谱，这是必要的定期收集光谱您调整针的位置，直到你能到您的记录相匹配的观测谱。质谱孔口从电针尖的距离将是1 - 5毫米。 </li><li> 外加电压：应用系统的电压会影响信号的离子强度和电Taylor锥稳定性。此外，最佳电压取决于你的电的解决方案和针尖形状。开始在你的一系列实验，确定最佳的频谱和信号稳定，产量为您的系统电压，然后所有后续的实验中使用的电压。对于我们的系统，适用于电压为2.0 - 5.0千伏提供最佳的信号强度和电稳定。在这个实验中使用2.5千伏。 </li></ol> 4。打开和调整SESI - MS分析<ol><li>首先确保电源电压关闭，该系统是电力出院。 1）确保电源电压指示灯熄灭，2）确保万用表上的电压为零，和3）接地的电气信息。 </li><li>安装适当的电解决方案，为您的实验。 </li><li>打开载气，并设定流量率适合你的实验。 </li><li>施加压力，以电水库启动电的解决方案交付的反应室。 </li><li>打开电源电压和电压调整到一个适当的值，为您的实验。 </li></ol>注意：此时离子源的金属表面，能够提供一个危险的冲击。周围的仪器工作时，一旦供电电压已打开，演习非常谨慎。 <ol start="6"><li>设置一个监测SESI - MS谱，而微调的调整施加电压的调整方法。使用的收购，你有你的系统和你的实验优化参数。清除多通道采集（MCA）“复选框（如果适用），每次扫描，产生一个独立的频谱，设置采集时间为10 - 15分钟，并开始收购。现在应该观察到载气的背景光谱。 </li><li>施加电压进行微调，调整，以获得稳定的总离子流色谱图（TIC），重现性好扫描相匹配的 CO 2为您以前的实验中扫描。一旦电压作了调整，继续收集5分钟，以确保仪器稳定的光谱和议会。 </li><li>一旦器乐稳定是保证，建立了适合于您的样品采集方法，调整的采集时间，数据的范围，和马华选择需要。收集记录的载体气体背景光谱。 </li></ol> 5。获取您的细菌培养的挥发性指纹<ol><li>为了收集一个空白频谱，直接通过旁路线的载气流量，并再附上空白​​样品（阀关闭）仪器的气体传输线。 </li><li>取样瓶，打开阀门，并关闭旁通线的阀。 </li><li>允许系统平衡为30秒，在这期间，在反应室中的湿度稳定。这一时期的平衡是获得重复性光谱的关键。为了确保该系统是平衡的，你不妨监察旅游业议会，这将改变在平衡期，稳定以后。 </li><li>一旦该系统是平衡的，启动频谱集合。 </li><li>频谱是收集后，首先开放的载气旁路线，然后关闭样品阀，并最后消除样品瓶中删除的取样瓶。嵌入式系统与载气为2 - 4分钟，除去从传输线的吸附水分和挥发物，防止样品，样品残留。 </li><li>重复步骤5.2 - 5.5每个细菌样本，间歇性地收取额外的空白光谱，以确保彻底空白减法。不完整的空白减法将导致在你处理的频谱化学背景山峰，是常见的大气压电离技术，外观（例如，邻苯二甲酸盐，有机硅，等）9 。 </li><li>收集您的光谱时，确保离子信号不超过线性检测仪器的限制，由议会和个别峰的最大强度确定。离子超过仪器的检测器上的限制，可以生成神器峰并不代表你的样品。 </li></ol> 6。代表性的成果作为SESI - MS谱的例子可为细菌性挥发物，正离子模式E.挥发指纹大肠杆菌和P.增长有氧24小时假单胞菌在LB -伦诺克斯在37 ° C时（图2）所示。的大肠杆菌大肠杆菌挥发频谱是由吲哚在m / z = 118为主，这给大肠杆菌大肠杆菌文化而P 的频谱特征的气味， 假单胞菌包含的protonatable峰较大的品种。 请注意，在动荡的光谱峰的相对强度取决于在第3节中所述的仪器参数。这些参数必须严格控制的实验，实验，以获得重复性光谱。 <img src="/files/ftp_upload/2664/2664fig2.jpg" alt="图2" /> 图2空白-减去正离子模式SESI - MS谱（20 - 150米/ Z）。 大肠杆菌大肠杆菌 K12和P.假单胞菌 PAO1挥发物LB -伦诺克斯有氧增长24 h后在37 ° C。 SESI光谱中观察到的峰的更多细节， 请参阅朱等8。

<ol>
	<li>Fuerstenau, S., Kiselev, P., Fenn, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESI-MS+in+the+analysis+of+trace+species+in+gases.">ESI-MS in the analysis of trace species in gases.</a> , (1999).</li><li>Wu, C., Siems, W. F., Hill, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+electrospray+ionization+ion+mobility+spectrometry/mass+spectrometry+of+illicit+drugs.">Secondary electrospray ionization ion mobility spectrometry/mass spectrometry of illicit drugs.</a> Anal. Chem. 72, 396-403 (2000).</li><li>Tam, M., Hill, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+electrospray+ionization-ion+mobility+spectrometry+for+explosive+vapor+detection.">Secondary electrospray ionization-ion mobility spectrometry for explosive vapor detection.</a> Anal. Chem. 76, 2741-2747 (2004).</li><li>Martinez-Lozano, P., Rus, J., de la Mora, G. F., Hernandez, M., de la Mora, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+electrospray+ionization+(SESI)+of+ambient+vapors+for+explosive+detection+at+concentrations+below+parts+per+trillion.">Secondary electrospray ionization (SESI) of ambient vapors for explosive detection at concentrations below parts per trillion.</a> J. Am. Soc. Mass Spectrom. 20, 287-294 (2009).</li><li>Martinez-Lozano, P., de la Mora, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-line+detection+of+human+skin+vapors.">On-line detection of human skin vapors.</a> J. Am. Soc. Mass Spectrom. 20, 1060-1063 (2009).</li><li>Martinez-Lozano, P., de la Mora, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+of+volatiles+in+breath.">Electrospray ionization of volatiles in breath.</a> Int. J. Mass Spectrom. 265, 68-72 (2007).</li><li>Martinez-Lozano, P., de la Mora, J. F., F, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+fatty+acid+vapors+in+breath+by+electrospray+ionization+and+atmospheric+pressure+ionization-mass+spectrometry.">Direct analysis of fatty acid vapors in breath by electrospray ionization and atmospheric pressure ionization-mass spectrometry.</a> Anal. Chem. 80, 8210-8215 (2008).</li><li>Guo, X. H., Bruins, A. P., Covey, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+typical+chemical+background+interferences+in+atmospheric+pressure+ionization+liquid+chromatography-mass+spectrometry.">Characterization of typical chemical background interferences in atmospheric pressure ionization liquid chromatography-mass spectrometry.</a> Rapid Commun. Mass Spectrom. 20, 3145-3150 (2006).</li><li>Rudzinski, C. M., Herzig-Marx, R., Lin, J., Szpiro, A., Johnson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathogen detection using headspace analysis. , 16-16 (2004).</li><li>Lechner, M., Fille, M., Hausdorfer, J., Dierich, M., Rieder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+bacteria+in+vitro+by+mass+spectrometric+fingerprinting:+A+pilot+study.">Diagnosis of bacteria in vitro by mass spectrometric fingerprinting: A pilot study.</a> Curr. Microbiol. 51, 267-269 (2005).</li><li>Schulz, S., Dickschat, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+volatiles:+The+smell+of+small+organisms.">Bacterial volatiles: The smell of small organisms.</a> Nat. Prod. Rep. 24, 814-842 (2007).</li><li>Ligor, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+methods+for+breath+investigation.">Analytical methods for breath investigation.</a> Crit. Rev. Anal. Chem. 39, 2-12 (2009).</li><li>Cao, W. Q., Duan, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breath+analysis:+Potential+for+clinical+diagnosis+and+exposure+assessment.">Breath analysis: Potential for clinical diagnosis and exposure assessment.</a> Clin. Chem. 52, 800-811 (2006).</li><li>Law, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+characterization+of+complex+viscous+liquids+at+the+molecular+level.">Rapid characterization of complex viscous liquids at the molecular level.</a> Angew. Chem. Int. Ed. 48, 8277-8280 (2009).</li><li>Wu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+analytes+from+cheese+products+for+fast+detection+using+neutral+desorption+extractive+electrospray+ionization+mass+spectrometry.">Sampling analytes from cheese products for fast detection using neutral desorption extractive electrospray ionization mass spectrometry.</a> Anal. Bioanal. Chem. 397, 1549-1556 (2010).</li><li>Fleming-Jones, M. E., Smith, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+organic+compounds+in+foods:+A+five+year+study.">Volatile organic compounds in foods: A five year study.</a> J. Agric. Food Chem. 51, 8120-8127 (2003).</li><li>Calkins, C. R., Hodgen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+meat+flavor.">A fresh look at meat flavor.</a> Meat Sci. 77, 63-80 (2007).</li></ol>

细菌产生的挥发物的不同组合，可利用 10-12细菌鉴定及代谢状况的评估。这里描述SESI - MS法提供了一种快速定性没有任何样品制备细菌挥发物（在两分钟或更少），产生细菌的“指纹”鉴定的品种在过去的几十年的其他8个大气压电离手段。 MS技术已应用于挥发性化合物的特性，包括选择性离子流管（SIFT）和质子转移反应质谱（PTR）。 SESI这些等离子的方法提供的独特优势在于，它是可能的特定片段峰（提供适当类型的质谱仪，已被改编为SESI），这是一个化合物鉴定的重要工具。我们没有解决在上面列出的协议的高峰碎片，但如何可用于细菌挥发物的定性碎片信息的例子，请参阅 朱 ，等。8 SESI - MS直接应用在原位检测细菌性肺部感染，通过呼吸分析，但也可以适用于任何设置挥发采样。例如，挥发物在尿液，血液，呼吸，这是相关的代谢紊乱的诊断分析，胃肠道疾病，癌症和环境接触，是非常适合SESI - MS 13,14 SESI - MS也有广泛的非临床VOC指纹识别应用程序，包括食品与成熟，老化，或破坏相关的特征挥发物的快速分析 15-18 。

<table border="1"><tbody><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td><td> 目录＃ </td><td> 评论 </td></tr><tr><td> API - 3000三重串联四极杆</td><td>仪器</td><td> SCIEX </td><td></td><td>分析师1.4.2（美国应用生物系统公司购买） </td></tr><tr><td> SESI离子源</td><td>仪器</td><td></td><td></td><td>定制;见参考文献6 </td></tr><tr><td>气体流量计</td><td>设备</td><td>科尔 - 帕默</td><td> EW - 03217 - 74 </td><td></td></tr><tr><td>二氧化碳</td><td>设备</td><td>公司Airgas </td><td>光盘I300 </td><td> ≥99.99％，纯</td></tr><tr><td>氮</td><td>设备</td><td>公司Airgas </td><td>倪UHP300 </td><td>超高纯度</td></tr><tr><td> 100毫升的玻璃媒体瓶</td><td>设备</td><td> VWR </td><td> 89012-114 </td><td> GL45螺丝螺纹</td></tr><tr><td>露儿端口的瓶盖</td><td>设备</td><td>生物化工射流</td><td> 00945T - 3 </td><td>第大会</td></tr><tr><td>露儿端口插头</td><td>设备</td><td>生物化工射流</td><td> 009LP </td><td>第大会</td></tr><tr><td>油管1 / 4“（外径）x 1 / 8”（编号） </td><td>设备</td><td>科尔 - 帕默</td><td> EW - 95875 - 02 </td><td>第组装及气体传输线</td></tr><tr><td>油管1 / 8“（外径）× 1 / 16”（编号） </td><td>设备</td><td>科尔 - 帕默</td><td> EW - 06605 - 27 </td><td>第大会</td></tr><tr><td>双向阀</td><td>设备</td><td>科尔 - 帕默</td><td> 07391-04 </td><td>第大会</td></tr><tr><td>过滤器，二级AAQ </td><td>设备</td><td> Balston过滤器</td><td> 9922-05 </td><td></td></tr><tr><td>甲酸，LC / MS级</td><td>试剂</td><td>费舍尔</td><td> A117 - 05AMP </td><td>电的解决方案</td></tr><tr><td>甲醇，LC / MS的级</td><td>试剂</td><td>费舍尔</td><td> A456 - 500 </td><td>电的解决方案</td></tr><tr><td>水，LC / MS的级</td><td>试剂</td><td>费舍尔</td><td> W6 - 500 </td><td>电的解决方案</td></tr></tbody></table>

characterizing bacterial volatiles using secondary electrospray ionization mass spectrometry (sesi-ms)

二次电喷雾电离质谱（SESI - MS），开​​发快速检测挥发性化合物的方法，而不需要样品预处理，一个。该方法最初是由芬恩和他的同事1，并已应用于检测非法毒品和爆炸物 3-4，皮肤挥发物 5的特性，并分析呼吸6-7。 SESI发生电离电的解决方案和挥发性待测之间的质子转移反应，因此适合杂有机分子的分析，就像在传统的电喷雾电离（ESI）。不过，与标准ESI，SESI质子转移过程发生在气相中的解决方案，而不是（图1），因此SESI最好是适合用于检测有机挥发物和气溶胶。 我们正在扩大使用SESI - MS检测细菌挥发物作为细菌的鉴定和表征 8方法。我们已经证明，SESI - MS挥发的指纹，与统计分析方法相结合，可以用来区分在生长介质的各种细菌属，种和混合文化，在这里，我们提供获得细菌使用SESI挥发指纹的步骤 - MS，包括应该优化，以​​确保强大的细菌的鉴定和表征的仪器参数。

Watch this Scientific Journal Video about Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS at JoVE.com

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

二次电喷雾电离质谱（SESI - MS），挥发性有机化合物（VOCs）的检测，而不需要任何样品预处理。该协议提供迅速细菌使用SESI - MS的挥发性有机化合物的特性（在几分钟之内）的说明。

使用辅助电喷雾质谱（SESI - MS）表征细菌挥发

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

characterizing-bacterial-volatiles-using-secondary-electrospray

Research

JoVE Journal

Bioengineering

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

17.9K 次观看.  University of Vermont. 二次电喷雾电离质谱（SESI - MS），挥发性有机化合物（VOCs）的检测，而不需要任何样品预处理。该协议提供迅速细菌使用SESI - MS的挥发性有机化合物的特性（在几分钟之内）的说明。

视频: Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

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

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

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

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

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

科研

生物工程

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

解吸电喷雾电离质谱成像的生物组织

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

全身质谱成像红外基质辅助激光解吸电喷雾电离（IR-MALDESI）

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

一种基于变形虫的快速图像细菌毒力检测方法

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...