JS is indebted to Prof. B. Hoge of the Inorganic Chemistry department, Bielefeld University, for the idea of establishing the presented inlet system. The analyzed phosphane was a generous gift from Prof. B. Hoge. Sample preparation of the analyzed compound was performed by M. Wiesemann. Photographs of the phospane were taken by Dr. J. Bader. The mechanical workshop of the faculty of chemistry is acknowledged for the manufacturing of the interface and the glass workshop of the faculty of chemistry for the manufacturing of the lockable test tubes with flanges. Prof. B. Hoge and Prof. H. Gr&#246;ger are acknowledged for funding of this publication.

1，样品制备<ol><li>使用定制的可锁定的试管有凸缘（ 图1），用于运输和样品转移到质谱仪。之前填充样品，疏散附着到多个歧管的Schlenk线的可锁定的试管，通过加热用热枪除去残留的水。泄试管用干燥氩气，并再次抽空，同时加热。 </li><li>沉浸在上锁的试管成冷阱充满液氮（注意:用液氮时要小心）。浓缩样品到从连接到的Schlenk线的歧管的样品容器的试管中，收在试管的顶部锁定以及歧管，并从氮浴中取出锁定试管。作为质谱分析是非常敏感的，有少量就足够了。大多数分析物会在分析过程中保留在可锁定试管和是可用于进一步的实验在测量后。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51858/51858fig1highres.jpg" width="500" /> 图用于样品转移1上锁的试管。 A）的法兰用于连接到冷进入系统中，试管（B）的特氟隆抽头，以使化合物的无空气的条件下运输，（C）的螺钉以操作特氟隆抽头。 2，测量质谱<ol><li>根据质谱仪（这里的制造商提供的说明来样测量，调整和校准质谱仪之前，一个高分辨磁质谱AutoSpec X（真空发生器，现在Waters公司，英国曼彻斯特）被使用。使用perflourokerosene（PFK）作为一个标准，调整质谱仪约2800 米处）的分辨率/ Z 119，10％峰谷定义。删除推从离子源的直接入口的杆和安装在试管的外接口（ 图2）。为了防止推杆头的加热，设置在入口的方法来在质谱仪中的控制软件"隔膜"。 </li><li>连接的可锁定的试管填充有样品到外接口的凸缘。打开外接口的针形阀和疏散口。抽真空后，小心地打开球阀，以离子源以完成抽真空步骤。关闭外接口的针形阀。注意:在测试管的特氟隆抽头具有在此步骤被关闭。 </li><li>在质谱仪中的软件开始质量测量。具有闭合针阀，打开试管的特氟隆抽头简略，使分析物的气相分子进入接口的外部部分。再次关闭铁氟龙水龙头。 注:该接口与TEFL之间的空间的外部部分一起关于抽头和可锁定的试管的凸缘（比较图1和图2B）作为分析过程中分析物的气体贮存器。 </li><li>同时观察离子源的真空测量仪小心打开针阀。这个步骤允许被分析物分子进入质谱仪的离子源。真空不应该在测量过程中下降到低于10 -5毫巴。 注:根据质量好的分析物的质谱的波动是在大约10 -6毫巴获得。该样品的质谱现在被记录。通常，试样量泄漏到仪器通过针阀足以记录质谱数分钟。的情况下的离子的强度降低，打开针阀多一点允许更多的采集时间。如果结果为70电子伏特的质谱的质量不令人满意，质谱可使用的低动能被记录电子， 例如 ，20伏特。 </li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51858/51858fig2highres.jpg" width="500" /> 图2冷进气系统与嵌合的VG高分辨磁质谱AutoSpec十甲离子源空上锁试管）可锁定的试管中，（B）的试管之间的法兰连接的冷进入系统，（C）的针形阀，（D）的不锈钢板与密封件作为连接到所述离子源中，直接入口（E）的界面中，推杆的陶瓷前端是可见的。 3，测量后<ol><li>关闭外接口的针形阀。关闭球阀至离子源。停止采集质谱中的软件。 </li><li>疏散进气系统而完全打开针阀。发泄球时的界面阀和针阀关闭。再次抽空界面，在该步骤中，以除去残留的样品的蒸气在界面打开针阀。重复此步骤，至少3倍。 </li><li>删除接口可锁定试管。继续进行下一个样品或来自离子源的凸缘移除外接口，它与推杆取代。 </li></ol>

<ol>
	<li>Field, F. H., Franklin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electron Impact phenomena and the Properties of Gaseous Ions Revised Edition. , (1957).</li><li>Schaeffer, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+Mass+Spectrometer+Ion+Source.">An Improved Mass Spectrometer Ion Source.</a> Rev. Sci. Instrum. 25, 660-662 (1954).</li><li>Yamashita, M., 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=Electrospray+Ion+Source+-+Another+Variation+of+the+Free-Jet+Theme.">Electrospray Ion Source - Another Variation of the Free-Jet Theme.</a> J. Phys. Chem. 88, 4451-4459 (1984).</li><li>Lubben, A. T., McIndoe, J. S., Weller, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+an+electrospray+ionization+mass+spectrometer+with+a+glovebox:+A+straightforward,+powerful,+and+convenient+combination+for+analysis+of+air-sensitive+organometallics.">Coupling an electrospray ionization mass spectrometer with a glovebox: A straightforward, powerful, and convenient combination for analysis of air-sensitive organometallics.</a> Organometallics. 27, 3303-3306 (2008).</li><li>Cooper, G. J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+Compositional+Control+in+{M12}+Cobalt+and+Nickel+Coordination+Clusters+Detected+Magnetochemically+and+with+Cryospray+Mass+Spectrometry.">Structural and Compositional Control in {M12} Cobalt and Nickel Coordination Clusters Detected Magnetochemically and with Cryospray Mass Spectrometry.</a> Angewandte Chemie International Edition. 46, 1340-1344 (2007).</li><li>Wang, W. S., Tseng, P. W., Chou, C. H., Shiea, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+1,2,3-hexatriene-5-one+monomer+by+low-temperature+atmospheric+pressure+ionization+mass+spectrometry.">Detection of reactive 1,2,3-hexatriene-5-one monomer by low-temperature atmospheric pressure ionization mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 12, 931-934 (1998).</li><li>Wang, C. H., 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=Detection+of+a+thermally+unstable+intermediate+in+the+Wittig+reaction+using+low-temperature+liquid+secondary+ion+and+atmospheric+pressure+ionization+mass+spectrometry.">Detection of a thermally unstable intermediate in the Wittig reaction using low-temperature liquid secondary ion and atmospheric pressure ionization mass spectrometry.</a> Journal of the American Society for Mass Spectrometry. 9, 1168-1174 (1998).</li><li>Eelman, M. D., Blacquiere, J. M., Moriarty, M. M., Fogg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shining+new+light+on+an+old+problem:+Retooling+MALDI+mass+spectrometry+for+organotransition-metal+catalysis.">Shining new light on an old problem: Retooling MALDI mass spectrometry for organotransition-metal catalysis.</a> Angewandte Chemie-International Edition. 47, 303-306 (2008).</li><li>Linden, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+injection+field+desorption+ionization:+a+new+tool+for+soft+ionization+of+samples+including+air-sensitive+catalysts+and+non-polar+hydrocarbons.">Liquid injection field desorption ionization: a new tool for soft ionization of samples including air-sensitive catalysts and non-polar hydrocarbons.</a> Eur. J. Mass Spectrom. 10, 459-468 (2004).</li><li>Gross, J. H., 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=Liquid+injection+field+desorption/ionization+of+reactive+transition+metal+complexes.">Liquid injection field desorption/ionization of reactive transition metal complexes.</a> Analytical and Bioanalytical Chemistry. 386, 52-58 (2006).</li><li>Peterson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometer+All-Glass+Heated+Inlet.">Mass Spectrometer All-Glass Heated Inlet.</a> Analytical Chemistry. 34, 1850-1851 (1962).</li><li>Stafford, C., Morgan, T. D., Brunfeldt, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mass+spectrometer+all-glass+heated+inlet.">A mass spectrometer all-glass heated inlet.</a> International Journal of Mass Spectrometry and Ion Physics. 1, 87-92 (1968).</li><li>Hayes, S. A., Berger, R. J. F., Mitzel, N. W., Bader, J., Hoge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorobis(pentafluoroethyl)phosphane:+Improved+Synthesis+and+Molecular+Structure+in+the+Gas+Phase.">Chlorobis(pentafluoroethyl)phosphane: Improved Synthesis and Molecular Structure in the Gas Phase.</a> Chemistry-a European Journal. 17, 3968-3976 (2011).</li><li>Zakharov, A. 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=Functionalized+Bis(pentafluoroethyl)phosphanes:+Improved+Syntheses+and+Molecular+Structures+in+the+Gas+Phase.">Functionalized Bis(pentafluoroethyl)phosphanes: Improved Syntheses and Molecular Structures in the Gas Phase.</a> European Journal of Inorganic Chemistry. , 3392-3404 (2013).</li></ol>

The authors have nothing to disclose.

从在标准样品制备程序，分解的化合物收购质谱载于本协议。所提出的技术被设计为金属organyls，硅烷和膦，这是很容易受到氧化和/或水解的分析，使得它有趣特别是对于无机化学家。为了达到最佳的效果，真空或空气的条件下在整个分析加以保存。因此该协议应认真遵守。的情况下，分析物进入与空气接触泄漏或错误处理的进口系统的，因为，造成严重后果如样品容器的爆炸可根据化合物的情况下进行分析（ 图4）。样品制备也应该只执行通过用于与的Schlenk线工作的，因为它们可以爆受过训练的人员由于不良的制造玻璃器皿真空下时。爆炸米飞行可以通过与空气因处理不当反应性化合物，在进入的Schlenk线的接触而引起的。液态氧的危险，在惰性气体中杂质冻结在寒冷的陷阱，也应考虑。液体氧的任何（上）有机化合物的接触可能导致引起的Schlenk线的爆炸剧烈反应。在分析过程中，在质谱仪的离子源中的压力应仔细观察，如在离子源的高压力可能会损坏仪器。 与挥发性金属organyls，极易受到氧化和/或水解硅烷和膦很少或根本不可能使用先进设备，最先进的与ESI或MALDI离子源，如根据一般进行样品制备和电离质谱分析大气条件。这里，冷进入系统呈现为物导入现有的方法的一个有价值的扩展。然而，冷入口系统是不与ESI和MALDI离子源兼容，因为它呈现气态分析物的离子化源​​的流动，而ESI需要的分析物溶液和MALDI分析物共结晶与基质的离子的生成的流程图。因为每个电离策略，EI也有一个缺点 - 由于电子束的能量高，碎裂频繁发生。取决于分析物的性质可能会导致在没有分子离子。然而，分析物可以使用碎片离子进行识别，特别是当所提供的化合物是高纯度的。用于明确识别，合成路线在此情况下是已知的。对于化合物，其中的分子离子是存在于质谱中，该片段离子可用于证实该分子的结构。由于冷入口系统的设计中，所提出的策略是有限的，以化合物具有一定的波动。分析物的波动性要足够，一llow的化合物在真空下在液氮温度下的转移 - 要么通过沸腾或升华。化合物与太低蒸气压不会产生高质量的质谱。单个质量测量之间的串扰很少观察到，并且可以通过仔细地抽进系统被进一步最小化。 在一般情况下，测量可以与每一个质谱仪配备有EI离子源和直接进样系统中进行。所呈现的冷进入系统是运行在几秒钟内，并且需要在质谱仪上没有改变。然而，冷进入系统的接口具有可定制的，根据所使用的EI质谱的现有入口的尺寸。根据可用的质谱仪的功能，附加的实验，可以进行诸如用于判定或确认的元素组成或精确质量测量质量分析离子显象管抽动能源法（MIKES）的裂解途径的阐明。因此，冷进气系统代表了一个额外的进样口的战略旁边用推杆在EI质谱经常使用的直接入口。

化合物，如金属organyls，硅烷，或phosphanes通过质谱法的分析并不总是可行的。几个这些化合物是已知的时，分解迅速与空气接触。因此，测量的质谱时，最重要的步骤是样品制备中，分析物的转移到质谱仪和离子发生在不存在空气。在这个协议中，我们描述了一种策略，以满足这些要求，并提出了一个入口系统，这使得它能够得到挥发性化合物以前没有被进行质谱分析的质谱图，由于其难以处理和快速分解的环境条件下。由此，明确识别新的或现有的挥发性金属organyls，硅烷和phosphanes，易被氧化或水解的，现在可以与质谱的协助下进行的。有两个要求其具有为了分析化合物必须满足这易于氧化或水解:在惰性条件下的样品制备和离子产生。最后的前提下可使用质谱仪的离子源在真空下操作很容易地得到满足。这与大部分基质辅助激光解吸/电离（MALDI）质谱，并与所有的电子轰击离子源（EI）质谱仪1,2的情况。电喷雾电离（ESI）是不为易被氧化或水解的化合物的分析很容易兼容，如在电离过程需要在环境条件3下的地方。然而，对于一些化合物，它们与氧或水，干燥，并与该最ESI源操作雾化气体的反应不大力是通过质谱法4足够用于分析。这也是对于电离策略类似于ESI， 例如 ，低温ESI，低温大气压电离和低温液体二次离子质谱SPECTR的情况下ometry 5-7。与此相反，样品制备和转移到惰性条件下的离子源是更有挑战性。两个MALDI和ESI文书已加上手套箱中以使在惰性气氛中4,8易于氧化和/或水解的化合物的样品制备方法。质谱仪接口到手套箱中任一个传输毛细管（ESI）或直接连接到手套式操作箱（MALDI）。液体喷射场解吸/电离（LIFDI） - -与敏感化合物的分析报告9,10的手套箱中，以经由传输毛细管质谱仪的耦合也将使用另一种离子化的策略是可能的。 此外，MALDI和LIFDI不适合于高挥发性化合物的分析。 MALDI需要的共结晶的分析物与基质和LIFDI需要被分析物上的沉积中的EMITTER从一个解决方案。与两个离子化策略是非常可能的是，分析物将蒸发随着溶剂。在对比MALDI仪器，EI质谱仪通常提供了几种方法用于引入样品进入离子源:直接导入探针（少量的固体颗粒，油，或蜡沉积到其使用推杆引入铝坩埚中） ，隔片入口（液体），或耦合用气相色谱仪。再次，在至少部分样品转移发生在环境条件下是难以在惰性气氛下进行。 在20世纪60年代，进样系统，提出了使样品的引入真空成的EI仪器的离子源-全玻璃加热进气系统（AGHIS）11,12。这里，将样品位于密封件的玻璃毛细管，将其插入到AGHIS内。接着，AGHIS抽真空和与所述样品的玻璃容器破裂。该AGHIS随后，加热以蒸发其中通过的泄漏装置达到的EI质谱仪的离子源的样品。当制备在手套箱内用样品的玻璃毛细管中，样品可以被引入到质谱仪中，没有任何接触到空气。然而，AGHIS是它不是市售的，难以组装，即使是熟练的吹玻璃车间的设备。由于直接入口之间的大尺寸切换使用推杆和AGHIS不是直线前进。 在我们的质谱实验室，我们在AGHIS的风格开发出了类似的进气系统。然而，由于这是不可能的加热进口系统中，分析物具有表现出以进入质谱仪的离子源有一定的波动。分析物的挥发性必须足够，以允许化合物在真空下在液氮德转印温度 - 无论是煮沸或升华。该定制进气系统包括一个不锈钢板，其被定位在直接进样系统，不锈钢管与针型阀，和一个凸缘，以可连接含有样品可锁定试管。冷进样系统的安装无需修改质谱仪（高分辨磁质谱AutoSpec X，真空发生器，现在Waters公司，英国曼彻斯特） - 冷进气系统和直接入口之间使用，可以在几秒钟内轻松完成推杆切换。 所呈现的进口系统是特别有用的，当金属organyls，硅烷，或phosphanes，易被氧化或水解，必须进行分析。这些化合物被用核谱核磁共振（NMR）或红外（IR）光谱法常用分析。不幸的是，这些方法允许并不总是一个明确的鉴定的化合物，因为它们产生incompletE资料， 例如 ，当元件如氯或溴是该分子的一部分。另一方面气体电子衍射可以提供关于分析物的详细信息，但是，该方法是非常耗时的，样品的制备是困难的，并且只有少数群体能够进行这些分析13,14。这里，对于金属organyls，硅烷，或phosphanes，易被氧化或水解被EI质谱分析中的冷进气系统是有机化学家能够明确识别的新型化合物大量使用对（上）由与信息提供他们关于特征碎片离子的分子和质量。为质谱的一种物质对测量的唯一先决条件是在减压一定的波动性。

<table border="0" cellpadding="0" cellspacing="0" width="971">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Name of Reagent/Material</td>
			<td style="width:71px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td style="width:435px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:311px;">VG Autospec X</td>
			<td colspan="2">Micromass Co. UK Ltd (now Waters)</td>
			<td style="width:435px;">other EI mass spectrometers with direct inlet using a push rod should also be compatible with this technique</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Lockable test tubes with flange</td>
			<td style="width:71px;">-</td>
			<td style="width:155px;">-</td>
			<td style="width:435px;">costum made, teflon tap should be used for locking the test tube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:311px;">Interface for lockable test tubes</td>
			<td style="width:71px;">-</td>
			<td style="width:155px;">-</td>
			<td style="width:435px;">costum made , interface is prepared from stainless steel. Needle valve has to be included into the interface-design!</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:311px;">Schlenk line</td>
			<td style="width:71px;">-</td>
			<td style="width:155px;">-</td>
			<td style="width:435px;">costum made, has to include vacuum pump for evacuation of thest tubes and cold trap with liquid nitrogen for trapping of the sample</td>
		</tr>
	</tbody>
</table>

analysis of volatile and oxidation sensitive compounds using a cold inlet system and electron impact mass spectrometry

这部影片提出了一个协议，用电子轰击电离挥发和氧化敏感的化合物的质量spectrometrical分析。挥发和氧化敏感的化合物通过质谱法的分析是不容易实现的，因为国家的最先进的所有质谱方法所需要的分析物（电喷雾电离），共中的至少一种的样品制备步骤， 例如 ，溶解和稀释与基质化合物（基质辅助激光解吸/电离），或所制备的样品转移到质谱仪的电离源分析物-crystallization，大气压的条件下进行。这里，使用进样系统的描述使用扇区字段质谱仪配备有电子轰击电离源挥发性金属organyls，硅烷，和phosphanes的分析。所有的样品制备步骤和样品引入的离子源质谱仪发生或者下无空气条件下或在真空条件下，使化合物的分析非常容易被氧化。所提出的技术是为无机化学家兴趣尤其，与金属organyls，硅烷，或phosphanes，它必须使用在惰性条件，如的Schlenk技术进行处理工作。操作的原理，提出在该视频。

的三（三氟甲基）膦的EI质谱示于图3中 ，化合物，其分解时迅速与空气（ 图4）接触。所呈现的界面允许质谱对这些化合物的直线前进的测量。新颖界面的操作是容易和快速的和操作的质谱仪，当与常规应用于直接入口使用推杆并没有任何障碍。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51858/51858fig3highres.jpg" width="500" /> 图3的三质谱（三氟甲基）膦， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51858/51858fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content宽度="5英寸"SRC ="/文件/ ftp_upload / 51858 / 51858fig4highres.jpg"WIDTH ="500"/&gt; 图4（A）贮存容器装满三（三氟甲基）膦。 （B）的容器填充有少量的三（三氟甲基）膦。 （C）在与空气接触，三（三氟甲基）膦点燃自发。 （照片礼貌J·贝德博士。）

Watch this Scientific Journal Video about Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry at JoVE.com

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

这部影片提出了一个协议，用电子轰击电离挥发和氧化敏感的化合物的质量spectrometrical分析。所提出的技术是为无机化学家兴趣尤其，与金属organyls，硅烷，或具有使用惰性条件下，例如在的Schlenk技术进行处理phosphanes工作。

挥发和氧化敏感的化合物使用冷进气系统和电子轰击质谱分析

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The ...

analysis-volatile-oxidation-sensitive-compounds-using-cold-inlet

Nanyang Technological University

Research

JoVE Journal

Chemistry

9.6K 次观看.  Bielefeld University. 这部影片提出了一个协议，用电子轰击电离挥发和氧化敏感的化合物的质量spectrometrical分析。所提出的技术是为无机化学家兴趣尤其，与金属organyls，硅烷，或具有使用惰性条件下，例如在的Schlenk技术进行处理phosphanes工作。 

视频: Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

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 ...

科研

化学

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

环境质谱可视化纹影摄影的使用

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

挥发性有机化合物与大气压化学电离四极杆质谱联用

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

寡 peptoids 的合成及质谱分析

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

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 ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

基于毛细管电泳的氢/氘交换，用于自上而下的质谱法对蛋白质进行构象表征

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...