Name: imaging of biological tissues by desorption electrospray ionization mass spectrometry
Uploaded: 2013-07-12T12:00:00.000+00:00
Duration: 6 min 21 s
Description: Watch this Scientific Journal Video about Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry at JoVE.com

支持这项工作是由ARRA NSF MRI仪器开发补助金＃0923179 FMF。我们感谢水族Asberry，帕克·佩蒂特生物工程研究所和生物科学组织学核心实验室协调员，协助组织切片。

1。组织切片<ol><li>商店闪光灯冷冻，在-80℃冷冻，直到整个组织准备切片。 </li><li>允许的组织样本中的温度达到之前的冷冻切片机切片（30分钟）。设置的叶片和样品温度至-30℃。 </li><li>一旦组织中已达到的温度，处理的试样用小钳子，切正面或背面的脑的部分的大脑还有足够的安装表面（ 即 ，如果前面的大脑是最重要的，大脑后部安装卡盘上）。 <ol><li>应用〜0.5毫升最佳切削温度化合物（华侨城，组织康樱）夹头中心，使用镊子，并保持样品在华侨城的地方，直到它变硬。华侨城一旦被完全冻结，安装夹头样品架。 </li><li>用于装载样品的OCT优选的最小量（对比试样安装在一个块中的OCT是常见的做在其他组织相容逻辑程序），以尽量减少样品的污染。样品的污染，由华侨城损害到DESI和成像过程中，由于离子抑制。 </li></ol></li><li>典型的组织切片所需的MS的成像范围从12至18微米厚。节组织的建议范围内所需的厚度。解冻挂载每个切片组织超过部分室温下的显微载玻片轻轻触摸。一旦样品被安装，请小心不要触摸，因为它会改变组织切片的化学分布和组成。 </li></ol>注意：我们建议安装两个部分每张幻灯片，使用一节进行优化，和其他成像。如果部分是没有立即成像，存储滑梯-80℃的冰箱在幻灯片中，直到准备好进行分析。 <ol start="5"><li>如果分析存储部分，从冰箱中删除幻灯片，立即转移到真空干燥器，并允许〜15-20最小解冻。离开试样干燥器中不再将干燥样品，，并降低DESI效率。一旦解冻，但是，在时间的关系，DESI离子源的优化工作将与组织样本，将样品解冻的期间作为一个理想的时间用于该设备的初始化。 <ol><li>使用作为DESI溶剂与1％乙酸的乙腈，打开取5μl/分钟的流速注射泵。较低的流速将降低的的DESI喷有效像素尺寸的影响点侧的，但更高的流速可能是必要的足够的灵敏度。因此，流量（通常1-5微升/分钟）应该为每个应用程序和所需的灵敏度和分辨率进行了优化。确保注射器在给定的流量的全面优化和成像包含足够的溶剂。 </li><li>一旦溶剂源尖端出现滴水，打开氮气雾化气体与PRESSURE 160磅。 </li><li>打开高压电源连接到离子源申请3,600 V。 </li></ol></li><li>装载的运动舞台上的幻灯片，并开始离子源和MS优化</li></ol> 2。 DESI优化<ol><li> DESI系统包括源探针（参见图2），注射器，泵或替代溶剂输送系统，压缩氮气，翻译样品台和安装。继续进行优化DESI源质谱仪​​一次已经调整为适当的质量范围和分析物。 <ol><li>如果源组件尚未尚未打开，请参阅第1.5.1-3的方向。 </li><li>这些部分中的所讨论的变量被推荐为生物组织成像。然而，这些变量进行优化，以不同类型的组织或样本。已报告的想象中的替代溶剂，流速，雾化气体的压力和电压的生物组织16-18纳克</li></ol></li><li>确保初期DESI源不接触载玻片上，而不是冲着任何部分组织部分。这将确保探头的初始优化过程中不被损坏，不会污染任何接触到的组织样本集的一部分。 <ol><li>一个很好的起点位置是从样品表面的前端3毫米到5毫米，从MS毛细管进样口，其中探头的角度55°相对于样品表面。内毛细血管的DESI探头应延长〜1毫米超越外毛细管所有这些参数将被优化</li></ol></li><li> MS接口毛细管应该有一个收集〜15°的角度相对于最佳的离子转移到样品表面。调整阶段高度，从而MS对样品表面的毛细管悬停在毛细管应该是小于1 mm的表面，尽量靠近不接触。 </li><li>对齐的DESI探头在x维度相对于向MS毛细管进样口，使它们直接符合。 </li><li>调整的y和z的的DESI源的最佳灵敏度，使用额外的组织切片的位置。注意DESI探针是针对一个样本区域，它将最终解吸和电离当这发生时，在这方面，信号的分析物将逐渐减小。重要的是尽可能快地执行此步骤。 <ol><li>因此，整个优化过程中，需要移动的喷射头的下面，以确保新鲜样品进行分析，然后在信号中的任何变化，由于源优化，而不是样品耗​​尽样品。然而，考虑到不同区域的组织的各种化学组合物的离子强度在整个组织切片，直接的比较，虽然不完全准确。丘脑的大脑提供一个相当大的大小的区域的更为一致的化学成分，但仍然没有完全统一。或者，标记红色的骗子，其中包含的染料若丹明6G（[M] +，米/ Z 443），在载玻片上相差的样品提供了一个强有力的MS响应由DESI也可以用于优化，但给出了不同的样品形式和分析物，这些条件可能仍然没有建立一个理想的生物组织相关。 </li><li>推荐Ÿ源尖和毛细管入口之间的分离：〜4毫米，推荐Ž分离针尖和样品表面：〜1.5毫米之间。虽然每个实验设置这些参数会略有不同。 </li><li>考虑到探头的几何形状和角度，以考虑到，y和z维度的位置，相对于溶剂和含被分析物的液滴的有效传输是相互关联的。在一个变量的改变将需要在后续的修改，以保持相同的喷流冲击位置和其传动角。 </li></ol></li><li>距离调整为从外最大的灵敏度，影响最小的光点尺寸的信号源毛细管内的毛细管挤出。注意：确保高电压是关闭，而这种调整。 </li><li>时观察到的最大信号源几何体将被认为是最优的。 <ol><li>第2.5-2.6优化的变量是相互关联的，因此，调整一个固有的需要进行重新调整，以维持必要的源样品入口的几何形状，或调整第1.5.1节中所讨论的条件3。 </li></ol></li><li>此外，加热元件周围的传输毛细管到质谱仪促进在电离过程中产生的带电分析物的液滴的去溶剂化，可以提高灵敏度。在这里，我们用绳子加热器盘绕在转让毛细管设置为100℃。 </li></ol> 3。布甲É成像<ol><li>在成像过程中，在样品台上将样品下方的的DESI探针和质谱入口毛细管和所有其他组件保持静止。舞台上的运动参数，应选择基础上的DESI影响羽规模和最佳的灵敏度。 <ol><li>较大的影响羽流将导致较高的离子强度，因为更多的样品被解吸，但是成像，它也将在更大范围内的像素尺寸和图像分辨率差从而导致。 </li></ol></li><li>本实验中使用的翻译阶段是建立和控制由LabView的程序，允许控制速度和给定尺寸的图像的行间距的光栅。本实验中使用的载物台的运动控制VI提供omnispect.bme.gatech.edu。另外，市售的的DESI源和阶段可以用于如2D自动化全方位喷雾源从Prosolia公司（印第安纳波利斯，IN USA）。 <ol><li>对于脑组织一典型的图像大小为10 x 15毫米，并使用阶段的运动参数，图像将需要约3小时。 <ol><li>编程LabView的VI或等效控制软件的期望的成像条件下，使用一个阶段的扫描速度为80微米/秒，和行线之间的间距为200μm。当使用的全喷雾源，运动参数应设置为100-200微米/秒的扫描速度和线间距为200μm。请注意，根据所希望的像素数在每个维度和必要的灵敏度，这些运动参数是可以改变的。 <ol><li>一个较小的线间距已被证明能提高图像质量，与扫描速度具有以下的效果。25，但要注意，这并不一定表明，提高了图像的分辨率，它是显着的，因为这是强烈地依赖于影响光斑大小。较慢的扫描速度和更小的行距将显着增加分析时间，因此必须优化作业捷思为每个不同的组织或样本。 </li></ol></li><li>与MS采集光谱扫描/秒，并作为1个像素/扫描产生的图象设定为1，阶段速度定义在x维度上的像素大小，行间距，而决定的像素大小中的y维度。虽然真正的像素的图像尺寸大于这是由于喷雾传播，速度较慢的运动允许更多的时间进行解吸和分析物的检测。 </li></ol></li><li>给目录期间要记录在LabVIEW VI图像的位置和时间的文件的路径和文件名。 </li></ol></li><li>在制备用于质谱数据采集，计算成像所需的总时间。本集团所用的Labview程序自动提供此值，但如果使用替代阶段控制，以此计算的MS数据采集的设置是必要的。 <ol><li>当使用全向自动喷雾源和阶段中，每行的图像获取在灵敏度独立运行。时间每行所需的给定的成像尺寸的行数指的是使用全喷雾要输入到质谱仪的软件的控制软件。 </li></ol></li><li>确保质谱仪的扫描速度为1频谱/秒，和设置在仪器的采集时间，以计算出的总时间相匹配。 <ol><li>设置光谱扫描速度为1频谱/秒。 </li><li>简介模式采集数据，并确保在m / z范围是适当的利益的物种。记住DESI也产生多电荷分析，在ESI。 </li><li>设置采集时间，以配合由LabVIEW图像总时间。 </li></ol></li><li>喷流冲击点在成像区域左上角的位置。 </li><li>同时开始的MS数据采集和舞台运动。 </li><li>数据采集​​结束后，返回到待机模式质谱仪。 </li><li>关闭高电压DESI源。 </li><li>转ØFF氮气。 </li><li>关闭注射泵。 </li></ol> 4。图像处理<ol><li>质谱数据，必须在结合阶段的时间和位置数据保存为一个文本文件通过LabView的“折叠”成2D图像相关联的坐标的像素与相应的光谱。这里呈现的图像处理采用基于Matlab软件是免费提供：omnispect.bme.gatech.edu。如果数据采集采用全方位喷雾源，萤火虫数据的转换软件是用来创建多维数据集的图像可视化在BioMAP（www.maldi msi.org）。 </li><li>所记录的色谱对于数据上JEOL AccuTOF。质谱仪收购，必须出口。CDF格式进行进一步的分析。 <ol><li>使用DataManager的内MassCenter，首先将采集的数据到centroided数据（工具→习得数据转换重心）。然后将数据导出。CDF格式通过使用键盘组合键Ctrl + Shift + E键FOL然后再由“工具”→“导出。 </li></ol></li><li>上传原料CDF质谱数据和两个文本文件，位置和时间，的OmniSpect网站。萤火虫处理的图像数据可以可视化BioMAP。 <ol><li>可以直接在网站上进行进一步处理的数据包括非负矩阵分解的统计分析或绘制一个单一离子的利益。 </li><li>另外，原始数据立方体可以导出在Matlab分析。 </li></ol></li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+mass+spectrometry+of+phospholipids.">Electrospray mass spectrometry of phospholipids.</a> Mass Spectrom. Rev. 22, 332-364 (2003).</li><li>Han, X., Holtzman, D. M., McKeel, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmalogen+deficiency+in+early+Alzheimer's+disease+subjects+and+in+animal+models:+molecular+characterization+using+electrospray+ionization+mass+spectrometry.">Plasmalogen deficiency in early Alzheimer's disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry.</a> J. Neurochem. 77, 1168-1180 (2001).</li><li>Murphy, E. J., Schapiro, M. B., Rapoport, S. I., Shetty, H. 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作者宣称，他们有没有竞争经济利益。

DESI源的几何形状的优化MSI实验成功的关键。促进的系统的对应的多个变量直接影响灵敏度和图像分辨率。如果在优化过程中，实验者获得信号有困难，我们建议使用Sharpie笔现货为基准，在幻灯片上绘制红色染料罗丹明6G，M / Z 443，在正离子模式，并产生一个强烈的信号，可用于初始优化。为DESI此外，溶剂的选择是至关重要的灵敏度，分析物传输和电离取决于从表面到所形成的薄膜的被分析物的?...

A correction was made to <a href="http://www.jove.com/video/50575" target="_parent">Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry</a>. There was an error with an author's name. The author's surname was appended with a missing character:
Rachel V. Bennett
instead of:
Rachel V. Bennet

Erratum: Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

无针对性的质谱成像，有利于发现和假说产生应用化学信息的收购。目标成像，在另一方面，还公知的化学，可以方便的灵敏度和选择性的增加，通过特定的方法的发展。质谱成像（MSI）是最常见的组织上进行二次离子质谱（SIMS），使用MALDI 2和环境离子化技术，包括解吸电喷雾电离（DESI），激光烧蚀电喷雾离子化（LAESI），4， 5和液体微结表面取样探头（LMJ-SSP）6 MALDI和SIMS，样品标本进行物理删除，并有平而薄，因为他们是在高真空下进行分析。 MALDI需要涂层的辐射吸收矩阵的样品，样品的制备增加一个额外的，繁琐的步骤。 SIMS具有最高的横向分辨率，但高度高能粒子轰击导致广泛的分子碎片。因此，微星环境的方法填补一个利基，用最少的样品制备软分析是可取的。然而，迄今为止，所有的方法仍然由平坦的样品表面的要求的限制。 DESI 7采用了气动辅助充电冲着样品表面解吸和电离分析物的溶剂喷雾解吸电离和后续DESI工作模型被称为“液滴回升模型”8-10主要液滴带电DESI探头所产生的碰撞的表面润湿，并形成薄膜，到其中的分析物被溶解的固-液微萃取机制8随后的液滴碰撞的结果中包含从表面提取的物质的二次液滴动量转移和起飞9,10，气体最终相离子被认为是离子蒸发，电荷残基模型或其他模型，11后通过ESI-状突起DESI然而，精确的离子形成过程中仍有待实验证明。的溶解度12的 DESI灵敏度是强烈地依赖于中的分析物的喷雾溶剂，解吸依赖于本地化的微萃取13 一起使用时，一个由软件控制的样品台，样品与车道步进DESI电离探针下方单向扫描，通过时域的m / z的信息是相关的化学物种的空间分布（ 图1）。 DESI-MSI原理实验报告由Van Berkel的凯尔泰斯在2006年以来的首次证明，14的技术已经相当成熟，15血脂分析，药物代谢产物3,16，17,18 disea报告应用硒的生物标志物，脑组织，19 3,18,20肺组织，肾组织18 18睾丸组织，18个肾上腺，17薄层层析板，21和藻类表面22例行DESI-MSI所获得的图像分辨率100-200微米，最终由喷雾提取的有效表面积，但已报告低至40微米的分辨率。23-25 ​​日这样的分辨率和便于分析，使DESI-MSI适合的快速和简单的分析生物组织样本的表面区域在0.5-5厘米2的范围内，使宝贵的空间信息的收购，以更好地理解生物过程26。在这里，DESI-MSI一个典型的应用程序作为一个例子，我们进行了成功的实验，涉及成像大鼠脑组织中脂质的程序细节。在协议中两个最重要的步骤是组织制备27和DESI离子源优化，如下所述。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura-Finetek</td><td>4583</td><td>&lt;a href=&quot;http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;amp;subcat=48&quot; target=&quot;_blank&quot;&gt;http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;amp;subcat=48&lt;/a&gt;</td></tr><tr><td>Acetonitrile</td><td>EMD</td><td>AX0156-6</td><td>OmniSolv, LC-MS Grade</td></tr><tr><td>Acetic Acid</td><td>Sigma Aldrich</td><td>695092-500 ml</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cryostat microtome</td><td>Thermo Scientific</td><td>CryoStar* NX70</td><td>Any available microtome can be used for tissue sectioning &lt;a href=&quot; http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;amp;groupType=PRODUCT&amp;amp;searchType=0&amp;amp;storeId=11152&amp;amp;from=search&amp;amp;ca=cryostar&quot; target=&quot;_blank&quot;&gt; http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;amp;groupType=PRODUCT&amp;amp;searchType=0&amp;amp;storeId=11152&amp;amp;from=search&amp;amp;ca=cryostar&lt;/a&gt;</td></tr><tr><td>Omni Spray&amp;#174;DESI Spray Head</td><td>Prosolia Inc.</td><td></td><td>Can also use the 2-D Omni Spray&amp;#174; Source kit instead of assembling components of imaging experiment &lt;a href=&quot; http://www.prosolia.com/sources.php&quot; target=&quot;_blank&quot;&gt; http://www.prosolia.com/sources.php&lt;/a&gt;</td></tr><tr><td>High Voltage Power Supply</td><td>Stanford Research Systems, Inc.</td><td>PS350/5000V-25W</td><td>&lt;a href=&quot;http://www.thinksrs.com/products/PS300.htm&quot; target=&quot;_blank&quot;&gt;http://www.thinksrs.com/products/PS300.htm&lt;/a&gt;</td></tr><tr><td>Rope heater, RTD, controller</td><td>Omega</td><td></td><td>&lt;a href=&quot;http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;amp;book=Heaters&quot; target=&quot;_blank&quot;&gt;http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;amp;book=Heaters&lt;/a&gt;</td></tr><tr><td>Labview</td><td>National Instruments</td><td>Version 7.1</td><td></td></tr><tr><td>Translational stage</td><td>Prior Scientific</td><td>Optiscan II</td><td>&lt;a href=&quot;http://www.prior.com/productinfo_auto_motorized_optiscan.html&quot; target=&quot;_blank&quot;&gt;http://www.prior.com/productinfo_auto_motorized_optiscan.html&lt;/a&gt;</td></tr><tr><td>AccuTOF Mass Spectrometer</td><td>JEOL</td><td>JMS-T100LC</td><td>Can use any mass spectrometer equipped with an extended capillary atmospheric pressure interface</td></tr></tbody></table>

imaging of biological tissues by desorption electrospray ionization mass spectrometry

质谱成像（MSI）提供不相关的分子调查在几百到几十微米规模的生物组织具有最高特异性和空间分辨率的信息。当在环境条件下，进行样​​品的前处理变得不必要了，从而简化了协议，而得到的信息保持高品质。解吸电喷雾电离（DESI）是一个基于喷雾环境的MSI的技术，它允许在露天的表面，即使是在体内直接采样。一起使用时，一个由软件控制的样品台，样品下方光栅DESI电离探针，通过时域的m / z的信息是相关的化学物种的空间分布。的DESI-MSI输出的保真度取决于源的方向和定位，对于样品表面和质谱仪入口。在此，我们回顾一下如何准备组织切片DESI我的maging和额外的实验条件下，直接影响图像质量。具体来说，我们描述成像大鼠脑组织切片的DESI-MSI的协议。

图3示出了代表性的频谱得到的未经处理的大鼠的脑切片。质谱在正离子模式，主要是由于其高离子化效率（归因于带正电的季铵基团）的磷脂酰胆碱。组织切片的总离子图像也显示在图3中 ，示出在整个脑切片的丰富的信号。通过文献比较表1主要血脂检测确定。 例如血脂的空间分布（ 图4）显示不同的卵磷脂物种的相对丰?...

Watch this Scientific Journal Video about Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry at JoVE.com

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

电喷雾解吸电离质谱（DESI-MS）是一个环境样本，包括生物组织，可以用最少的样品制备成像方法。通过离子探针光栅下面的示例，这个喷雾基于技术组织切片内辨别分子功能提供了足够的空间分辨率。

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 ...

imaging-biological-tissues-desorption-electrospray-ionization-mass

Research

JoVE Journal

Bioengineering

18.5K 次观看.  Georgia Institute of Technology. 电喷雾解吸电离质谱（DESI-MS）是一个环境样本，包括生物组织，可以用最少的样品制备成像方法。通过离子探针光栅下面的示例，这个喷雾基于技术组织切片内辨别分子功能提供了足够的空间分辨率。

视频: Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

科研

生物化学

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 ...

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 ...

生物工程

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

使用循环离子淌度光谱仪进行串联离子淌度实验

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

化学

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

常压LAESI质谱生物组织和生物膜的分子成像

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

生物学

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

单探头的应用:根据环境条件质谱成像和单细胞分析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI

The medicinal use of traditional Chinese medicine is mainly due to its secondary metabolites. Visualization of the distribution of these metabolites has become a crucial topic in plant science. Mass spectrometry imaging can extract huge volumes of data and provide spatial distribution information about these by analyzing tissue slices. With the advantage of high throughput and higher accuracy, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is often used in biological research and in the study of traditional Chinese medicine. However, the procedures used in this research are complicated and not affordable. In this study, we optimized sectioning and DESI imaging procedures and developed a more cost-effective method to identify the distribution of metabolites and categorize these compounds in plant tissues, with a special focus on traditional Chinese medicines. The study will promote the utilization of DESI in metabolite analysis and standardization of traditional Chinese medicine/ethnic medicine for research-related technologies.

In this study, a series of methods are presented to prepare DESI-MSI samples from plants, and a procedure of DESI assembly installation, MSI data acquisition, and processing is described in detail. This protocol can be applied in several conditions for acquiring spatial metabolome information in plants.

使用DESI-MSI鉴定中药空间代谢组中代谢物的可视化

The medicinal use of traditional Chinese medicine is mainly due to its secondary metabolites. Visualization of the distribution of these metabolites has ...

医学

MALDI-TOF Mass Spectrometry

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing compounds in the gaseous state, samples are embedded in a matrix, which is struck by a laser. The matrix absorbs the majority of the energy; some of this energy is then transferred to the sample, which ionizes as a result. Sample ions can then be identified using a time-of-flight analyzer (TOF).
This video covers principles of MALDI-TOF, including matrix selection and how TOF is used to elucidate mass-to-charge ratios. This procedure shows the preparation of a MALDI plate, the loading of samples onto the plate, and the operation of the TOF-mass spectrometer. In the final section, applications and variations are shown, including whole-cell analysis, characterization of complex biological samples, and electron spray ionization.
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Matrix-assisted laser desorption ionization, or MALDI, is a mass spectrometry ion source ideal for the analysis of biomolecules. Most ion sources remove structural information from large, fragile biomolecules. MALDI maintains structural integrity, and therefore information, while accelerating the molecules into the mass analyzer, which separates the compounds based on size and charge. The most commonly coupled with MALDI is the time of flight, or TOF, mass analyzer. This video will show the concepts of MALDI ionization, a general procedure, and some of its uses in biochemistry.
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For mass spectrometry to function, molecules must be ionized into the gaseous state. In MALDI, the sample is embedded in a matrix, typically an organic compound containing aromatic and conjugated double bonds.
When a laser pulse strikes this mixture the matrix absorbs the majority of the energy, rapidly heats, and is desorbed, or released, from the surface. The energized matrix transfers some of its energy to the biomolecules, desorbing and then ionizing them.
MALDI is typically paired with a time of flight, or TOF, mass analyzer. An electric field applies kinetic energy to the ions, moving them into a field-free region called a drift tube. The velocity of the ions as they move through the tube is related to their mass-to-charge ratio, so heavier particles travel slower through instrument. A detector at the end of the tube measures each ion&#39;s flight time. With this knowledge, as well as the tube length and applied field strength, the mass-to-charge ratio of each ion can be elucidated.
This plot of signal intensity to mass-to-charge-ratio, known as a mass spectrum, can be compared to a library of collected spectra. If no matches are found, it can molecules can be identified by further techniques, such as tandem mass spectrometry. For more information, see this collection&#39;s video on the topic.
Now that the basics of MALDI-TOF have been discussed, let&#39;s look at the process in the laboratory.
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Before beginning an experiment, it&#39;s important to consider the choice of matrix from which samples will be desorbed. It must absorb the laser energy, be stable in a vacuum, not react with the target molecules, and be able to desorb. Depending on the sample, different matrices are preferred. For a large protein, a combination of CHCA and DHB has shown better separation of the peaks, called resolution, than the individual matrices.
There are a number of ways to prepare samples. We&#39;ll show what is known as the &#34;double-layer&#34;, or &#34;sandwich,&#34; method. To begin, clean the MALDI plate with ultra-pure reagents, as mass spectrometry is very sensitive to contamination. Dry the plate with a stream of inert gas.
Next, a saturated matrix solution is made, typically with an organic solvent . The solution is streaked onto the MALDI plate and dried. A second saturated solution of matrix containing trifluoroacetic acid, or TFA, is prepared. TFA helps ions into the gaseous phase.
Next, the sample solution is added on top of the dried matrix spot. Add the matrix solution containing TFA on top of the sample, thereby completing the matrix &#34;sandwich&#34;. Homogeneity of the spot can be verified under a low-powered microscope.
Plate a calibration standard, which is a mixture with a wide range of known masses and is used to correlate the time-of-flight to m/z. Finally, plate the matrix alone as a negative control.
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To analyze the spots, place the target plate into the instrument. Ensure there&#39;s no debris present, allowing for the formation of a tight vacuum. In the software, select the standard, negative control, and samples of interest. Label the spots with the correct identification.
The ion source and lens voltages can be manipulated to improve performance of the analysis. This will depend on the specifics of the instrument and sample. Focus on the standard spot and calibrate the instrument with the software.
Next, collect spectra from each of the sample spots. Try a few different locations on the spot to maximize the quality of the collected data. Once finished, the MALDI plate can be collected and reused after cleaning.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the ways MALDI is utilized, and a different ionization technique.
In addition to biomolecules, MALDI can be used to analyze living cells. Macrophages are immune cells that take on one of several different forms, based on their microenvironment. After exposing the cells to various signaling molecules, or cytokines, they can be added directly to the plate, and analyzed. The MALDI spectra can be used as unique &#34;fingerprints&#34;, depending on the cytokine used.
Complex biological samples like mammalian sebaceous secretions require a step of purification before MALDI analysis. Thin layer chromatography is one such technique that relies on the components&#39; polarity. The compounds are collected from the TLC pate, purified, and transferred to a MALDI matrix. The resulting spectra verify the identity and purity of the separated biomolecules from the mammalian sebaceous secretions.
Another common ion source for biomolecules is electrospray ionization, or ESI. In this method, the sample is injected into the instrument, where a high voltage is applied, creating an aerosol of charged droplets. As the solvent in the droplet evaporates, the charge is moved to the sample molecules, till they are completely gaseous. ESI doesn&#39;t require the spotting procedure, and the sample can be injected directly into the instrument. On the other hand, ESI is more sensitive to the presence of buffer components and other contaminants, meaning MALDI is more robust.
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You&#39;ve just watched JoVE&#39;s video on MALDI mass spectrometry. This video described the theory behind the instrument, went over a general procedure, and covered some of the uses of the technique. Thanks for watching!
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MALDI-TOF Mass Spectrometry-Principle, Operation and Uses

MALDI TOF 质谱

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing ...

教育

JoVE Science Education

Electrospray Ionization (ESI) Mass Spectrometry

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional electron impact ionization methods. Accordingly, electrospray ionization (ESI) is the favored method for vaporizing and ionizing biomolecules as it circumvents rapid fragmentation and enables the recording of mass signals for the entire biomolecule.
ESI utilizes electrical energy to transfer ions from the liquid phase of the sample into the gaseous phase. The analyte biomolecule is premixed with an ionic liquid&#8212;a nonmolecular compound serving as the ionization source. The solution is sprayed through a high-voltage capillary to generate a fine aerosol. The voltage difference applied between the capillary and the entrance of the analysis chamber causes the movement of charged droplets toward the analysis chamber. The ionic liquid from the aerosol evaporates during this movement, leaving behind a variably protonated or ionized analyte molecule. These charged species are detected in the mass spectrum at an m/z value equivalent to the sum of the analyte molecule&#39;s molecular mass and the ions&#39; molecular weight from the ionic liquid associated with the analyte molecule.
The position of mass signals varies depending on the type of cations present in the ionic liquid. For example, the presence of sodium ions in the ionic liquid leads to an (M+23) peak in the mass spectra, while the presence of a proton results in an (M+1) peak.

电喷雾电离 (ESI) 质谱法

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional ...