The authors thank Professor H. Troy Ghashghaei from NCSU Department of Molecular Biomedical Sciences for providing the whole mouse tissue. The authors also gratefully acknowledge the financial assistance received from National Institutes of Health (R01GM087964), the W.M. Keck foundation, and North Carolina State University.

注:以下协议描述的所有执行IR-MALDESI MSI实验的必要步骤。深入可以在其他地方5,6-找到关于红外MALDESI源的优化几何形状及其与激光，阶段同步和质谱仪的信息。在这个协议中使用的动物组织样品按照机构动物护理和使用委员会（IACUC）和北卡罗莱纳州立大学的规定取得。 1.组织准备<ol><li>通过在通风橱中放置〜200毫升异戊烷在干净的烧杯干冰的次级容器内制备异戊烷/干冰浴。在任何时候都处理异戊烷/干冰浴和组织时，使用防护手套和护目镜。 <ol><li>通过安乐死过量溴乙醇2日龄整个新生小鼠小狗（7.5毫克/克体重），然后冻结组织中的异戊烷/干冰浴保持组织结构。使用一个预清洁编对镊子来放置，并从异戊烷/干冰浴移开鼠标小狗中cryomold。在-80℃直至分析存储快速冷冻组织。 </li></ol></li><li>使用防护手套，应用最佳切割温度（OCT）安装介质的层至40mm的低温恒温器试样圆盘。冷冻整个鼠标轻轻放置到华侨城涂层试样架坚持鼠标光盘在所需方向进行切片。 注意:OCT用于仅粘附的组织，以在试样圆盘用于切片。不要完全嵌入组织中OCT和华侨城是已知有MSI分析不利影响避免过剩。其他媒体，例如明胶，可用于冷冻嵌入组织切片它9之前。 </li><li>放在珀尔帖样品架光盘（与华侨城和组织切片）的低温恒温器，按佩尔蒂埃电源按钮。等待10分钟样品来热平衡。 </li><li>放置标本盘（与组织安装在它）该盘片保持器内，并面对组织向下到用于分析期望的平面。切片组织成使用容纳在低温恒温器10内的旋转式切片机在-20℃下所需厚度的部分。 注意:对于全身分析，切片组织成25微米厚的部分，以保持组织完整性。为更小的区域（ 例如 ，肝，脑，肾），切片组织成10微米厚的切片。 <ol><li>使用防倾玻璃板，以防止切片组织切片从滚动。使用低温恒温器真空软管和刷子去除不需要的组织碎片。一旦组织切片，用刀片护罩覆盖锋利的切片机刀片，以免造成人身伤害。 </li></ol></li><li>对试样板东方鼠部分和通过使滑动尽可能靠近于组织部分解冻贴装到预清洗的玻璃显微镜玻片，而不触及它10。 注:对于定量MSI experiments，涂层用自动气动喷雾器安装所述组织之前，与内部标准的幻灯片，和解冻贴装上涂布的载玻片11上的组织切片。 </li><li>删除习惯使用刀片喷射器切片组织切片机刀片，并安全地丢弃在适当的"锐利废物"容器中的刀片。 </li><li>保持低温恒温器内部的载玻片，直到步骤3.2，以保持组织的冷冻保存。 </li></ol> 2.红外MALDESI准备/校准<ol><li>打开中红外激光发射计算机上的激光控制中的应用。选择使用由制造商提供的激光控制应用所需的波长。红外MALDESI实验在2940纳米下进行。 注:最近的实验研究中红外的激光波长的依赖和冰矩阵这似乎是具体 ​​的分析指出8差异。 </li><li>打开雄鹿E控制器，启动自定义用户控制程序（RASTIR），和校准使用home键的舞台上的地位。 </li><li>准备电的解决方案。通常情况下，使用以50:50的比例（体积/体积）甲醇/水含0.2％甲酸在正离子模式电喷雾溶剂。根据实验选择电溶剂组合物。例如，使用5毫米氢氧化铵在负离子模式成像应用电改性剂。 注意:对于极性转换红外MALDESI微星，使用1mM的乙酸作为改性剂。这种改性剂被发现最适合于在两个正，负离子模式12获得稳定的和可重复的信号。 </li><li>填充1ml注射器具有电溶剂，并冲洗用新溶剂中的二氧化硅毛细管。 </li><li>使用源的参数，如:ESI-光点距离对齐ESI发射极上轴与MS入口，点进距离，样品的高度，并溶剂流速已使用统计DO优化<html> E代表组织切片6的分析。注:参见图1示意描绘这些参数的优化值组织切片的MSI。 </li><li>启动电和评价通过监测总离子电流（TIC）其中，在这一点上，仅由环境化合物其稳定性（&gt; 10分钟）。典型地，&lt;10％以上10〜15分钟的TIC变化是稳定的良好指示。 </li></ol><img alt="图1" src="/files/ftp_upload/53942/53942fig1.jpg" /> 图1. IR-MALDESI原理和参数。（A）IR-MALDESI源设置（不按比例）和可调节参数的示意图。 （ 二 ）优化参数值组织切片成像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53942/53942fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 冰阵标题"&gt; 3。沉积<ol><li>注意:将样品在舞台前，关闭电。 </li><li>放置解冻上的组织到摄像机视野内的红外MALDESI样品板。 </li><li>确保手是明确ESI发射器并重新启动电。 </li><li>关闭红外MALDESI源和净化机箱的访问门用干燥的氮气，以防止水凝结所述组织的表面上。一旦外壳到达内部的相对湿度&lt;3％，打开直流电源（〜12V）到珀耳帖板，其将冷却所述样品板。注意:在珀耳帖冷却阶段的冷却过程是在别处详细13讨论。 </li><li>冷却阶段-9℃，并允许上的组织，以达到热平衡（〜5-10分钟）。 </li><li>停止氮气吹扫，并且通过打开源接入门暴露组织的环境相对湿度。冰的薄层将沉积在共珀尔帖阶段的LD表面和安装组织部分由于来自空气的水凝华。 注意:如果在实验室的相对湿度低于10％-15％，将温水的烧杯中的外壳内以促进冰基质层的形成。 </li><li>形成在冰基质层后，关闭检修门，并重新启动干氮气的流动，以相对湿度降低至10±2％。调整氮气流量，以保持相对湿度在这个水平上，经验上发现的冰的形成和升华的平衡整个实验6保持冰的恒定层。 </li></ol> 4.质谱成像数据采集<ol><li>使用RASTIR GUI（ 如图2），点击"LASER位置"按钮，移动舞台分析位置。使用激光的调整二极管以确保离组织区域将被消融。火场外的组织重新激光射击祗园校准激光相对于摄像机视图所抵消。点击"激光测试火"按钮，更新激光位置。 </li><li>返回到"相机位置"，并通过将激光对准十字线的激光烧蚀点（ 图2-1）更新RASTIR激光位置。 </li><li>点击感兴趣区域（ROI）按钮的区域和调整ROI的大小和位置的组织切片被分析（ 图2-2）。输入相应的实验参数，如X和Y方向（单位mm）（ 图2-3）步长，并命名在"项目名称"框（ 图2-4），MSI文件。 <ol><li>当绘制ROI，包括了组织，充当控制的一部分。使用高峰采摘这一关，组织切片（步骤5.4）。 注意:在组织中的激光的解吸直径（点尺寸）大约为150微米;然而，更高的空间分辨率可以通过USI获得纳克过采样方法14,15。 </li></ol></li><li>选择从下拉框中，每像素的激光脉冲（整数值）的数目，以及激光触发器和质谱仪信号采集（ 图2-5）之间的延迟的适当的激光频率。通常使用每像素两个激光脉冲在20赫兹到在IR-MALDESI微星实验完全解吸材料，具有10毫秒的延迟时间，以允许产生到达质量分析器测量5离子。 注:选择最高的重复率使激光能够在操作。这是最近表明，使用较高的重复率将提高分析物探测16。 </li></ol><img alt="图2" src="/files/ftp_upload/53942/53942fig2.jpg" /> 图2.用户界面IR-MALDESI MSI操作。在RASTIR扫描控制程序的截图呈现。对于PERF的步骤orming一个微星实验是:（1）定位的激光点，（2）绘制的ROI，（3）在选择阶段步长大小（单位mm），（4）赋予的名称到该文件，（5）选择正确的数每个像素脉冲与所需的重复率，（6）检查列表成像和MS设置沿。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53942/53942fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li>选择质谱仪的参数，如电离模式下，电压，溶剂流速，毛细管温度，并在质谱仪软件喷射时间。参考表1对在全身的IR-MALDESI微星使用的参数的一个例子。 注意:由于在生物样品和缺乏微星分析分离方法的复杂性，对IR-MALDESI源耦合到一个高分辨力和高的质量准确度的仪器。采集方法可以加载analysES如平行反应监测（PRM）7或极性切换MSI 12。 </li><li>一旦所有参数（激光，舞台，和质谱仪）已被选定，将在MS同步"握手"模式，并启动MS采集。 </li><li>使用RASTIR成像软件，验证所有步骤清单（ 图2-6）通过检查框完成，加载程序。一旦程序被加载，"运行"按钮将变为可用。按Run启动MSI信号采集。 </li><li>一旦成像实验完成后，停止质谱仪采集并放置在待机状态下在仪器。关闭氮气吹扫到机箱，关掉电源，珀耳帖板，并关闭级控制器和激光。 </li><li>打开检修门，从源头机箱内取出电发射头，并使用防护手套删除扩展加热的金属质谱入口。注意:扩展<html>金属质谱入口会很烫。 </li><li>戴手套和护目镜，清洗在15％的硝酸经超声波浴中的扩展金属毛细管入口10分钟，然后在HPLC级水10分钟，最后在HPLC级甲醇10分钟。擦干金属毛细管入口在氮气流中，并重新插入MS。注:在适当的废物容器后处置溶剂。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="432"><colgroup><col span="2" /></colgroup><tbody><tr height="22"><td dir="LTR" height="22" style="height:22px;width:216px;"> 参数 </td><td dir="LTR" style="width:216px;"> 值 </td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">电离模式</td><td dir="LTR" style="width:216px;">正面的</td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">电电压</td><html> R"的风格="WIDTH:216px;"&gt; 3.8-4.2千伏</td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">溶剂流速</td><td dir="LTR" style="width:216px;"> 2微升/分钟</td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">毛细管温度</td><td dir="LTR" style="width:216px;"> 275℃ </td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">扫描范围</td><td dir="LTR" style="width:216px;"> M / Z 250-1000 </td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">扫描类型</td><td dir="LTR" style="width:216px;">全扫描</td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">注射时间</td><td dir="LTR" style="width:216px;"> 110毫秒</td></tr><tr height="22"><td dir="LTR" height="22" style="height:22px;width:216px;">解析力</td>宽度:216px;"&gt; 14万</td></tr></tbody></table>在全身IR-MALDESI微星采用表1.仪器参数。 5.数据分析<ol><li>由仪器生成的原始数据文件转换为数据格式，如mzXML 17或18 imzML使用免费软件如MSConvert 17和imzML转换器19。 </li><li>开始MSiReader V1.0，高分辨力MSI数据20的分析制定，有专门的处理计算机上的开源软件。加载映像文件到MSiReader。对于MSiReader的用户界面和它的一些内置的功能，参见图3。 </li><li>通过输入其M / Z生成的目标分析物离子地图，选择百万分之适当的M / Z窗口（PPM）或汤姆森（TH）（ 图3-1）。对于高分辨率（RP）仪器选择在低ppm范围内的一个窗口。 注:将合适的M / Z窗口取决于仪器即IR-MALDESI源耦合到的RP和质量的测量精度（MMA）。 </li><li>进一步解释使用内置的功能，如插补（ 图3-2），标准化（ 图3-3），光学图像叠加（ 图3-4），和峰值采摘（ 图3-5）20的数据。 </li></ol><img alt="图3" src="/files/ftp_upload/53942/53942fig3.jpg" /> 图3. MSiReader的用户界面; V1.0 20。一旦一个文件被加载到软件中，感兴趣分析物的离子映射由（1）输入ppm或钍的M / Z和公差显示。还可以执行诸如（2）内插或（3）归一化进一步分析。组织的光学图像也可以导入并叠加离子的地图（4）为更好的可视化。对于不相关的分析峰的拾取功能（5）可用于通过选择组织（品红色线）的区域和参考区域外组织（绿盒）提取组织特异性的峰值。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53942/53942fig3large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <ol start="5"><li>使用峰拾取函数生成使用用户定义的标准组织特异性m / z值的列表。注:PeakFinder算法使用两个区域平均质谱差异。然后，这些m / z值可以搜索针对代谢物数据库如METLIN 21或脂质MAPS 22 <ol><li>参考图3为选择一个询问组织区域（品红多边形ROI），并关断组织的参考区域（绿色多边形ROI）的一个例子。 </li></ol></li></ol>

<ol>
	<li>Mcdonnell, L. A., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry.">Imaging Mass Spectrometry.</a> Mass Spectrom. Rev. 26, 606-643 (2007).</li><li>Chughtai, K., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+imaging+for+biomedical+tissue+analysis.">Mass spectrometric imaging for biomedical tissue analysis.</a> Chem. Rev. 110 (5), 3237-3277 (2010).</li><li>Robichaud, G., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Mass+Spectrometry+Imaging.">Atmospheric Pressure Mass Spectrometry Imaging.</a> Encycl. Anal. Chem. , (2014).</li><li>Sampson, J. S., Hawkridge, A. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+detection+of+multiply-charged+peptides+and+proteins+by+matrix-assisted+laser+desorption+electrospray+ionization+(MALDESI)+Fourier+transform+ion+cyclotron+resonance+mass+spectrometry.">Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry.</a> J. Am. Soc. Mass Spectrom. 17 (12), 1712-1716 (2006).</li><li>Robichaud, G., Barry, J. A., Garrard, K. P., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+imaging+source+coupled+to+a+FT-ICR+mass+spectrometer.">Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging source coupled to a FT-ICR mass spectrometer.</a> J. Am. Soc. Mass Spectrom. 24 (1), 92-100 (2013).</li><li>Robichaud, G., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IR-MALDESI+Mass+Spectrometry+Imaging+of+Biological+Tissue+Sections+Using+Ice+as+a+Matrix.">IR-MALDESI Mass Spectrometry Imaging of Biological Tissue Sections Using Ice as a Matrix.</a> J. Am. Soc. Mass Spectrom. 25 (3), 319-328 (2014).</li><li>Barry, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+Antiretroviral+Drugs+in+Tissue+by+IR-MALDESI+MSI+Coupled+to+the+Q+Exactive+and+Comparison+with+LC-MS/MS+SRM+Assay.">Mapping Antiretroviral Drugs in Tissue by IR-MALDESI MSI Coupled to the Q Exactive and Comparison with LC-MS/MS SRM Assay.</a> J. Am. Soc. Mass Spectrom. 25 (12), 2038-2047 (2014).</li><li>Rosen, E. P., Bokhart, M. T., Ghashghaei, H. T., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Desorption+Conditions+on+Analyte+Sensitivity+and+Internal+Energy+in+Discrete+Tissue+or+Whole+Body+Imaging+by+IR-MALDESI.">Influence of Desorption Conditions on Analyte Sensitivity and Internal Energy in Discrete Tissue or Whole Body Imaging by IR-MALDESI.</a> J. Am. Soc. Mass Spectrom. 26, 899-910 (2015).</li><li>Nelson, K. A., Daniels, G. J., Fournie, J. W., Hemmer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+whole-body+zebrafish+sectioning+methods+for+mass+spectrometry+imaging.">Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging.</a> J. Biomol. Tech. 24 (3), 119-127 (2013).</li><li>Park, J. J., Cunningham, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+sectioning+of+slice+preparations+for+immunohistochemistry.">Thin sectioning of slice preparations for immunohistochemistry.</a> J. Vis. Exp. (3), e194 (2007).</li><li>Bokhart, M. T., Rosen, E., Thompson, C., Sykes, C., Kashuba, A. D. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry+imaging+of+emtricitabine+in+cervical+tissue+model+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization.">Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization.</a> Anal. Bioanal. Chem. 407 (8), 2073-2084 (2015).</li><li>Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+Switching+Mass+Spectrometry+Imaging+of+Healthy+and+Cancerous+Hen+Ovarian+Tissue+Sections+by+Infrared+Matrix-Assisted+Laser+Desorption+Electrospray+Ionization+(IR-MALDESI).">Polarity Switching Mass Spectrometry Imaging of Healthy and Cancerous Hen Ovarian Tissue Sections by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI).</a> Analyst. 141, 595-605 (2016).</li><li>Hsu, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Application+of+a+Low-Temperature+Peltier-Cooling+Microscope.">Design and Application of a Low-Temperature Peltier-Cooling Microscope.</a> J. Pharm. Sci. 85 (1), 70-74 (1996).</li><li>Jurchen, J. C., Rubakhin, S. S., Sweedler, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-MS+imaging+of+features+smaller+than+the+size+of+the+laser+beam.">MALDI-MS imaging of features smaller than the size of the laser beam.</a> J. Am. Soc.Mass Spectrom. 16 (10), 1654-1659 (2005).</li><li>Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular-level+mass+spectrometry+imaging+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+by+oversampling.">Cellular-level mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) by oversampling.</a> Anal. Bioanal. Chem. 407 (8), 2265-2271 (2015).</li><li>Rosen, E. P., Bokhart, M. T., Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+C-Trap+Ion+Accumulation+Time+on+the+Detectability+of+Analytes+in+IR-MALDESI+MSI.">Influence of C-Trap Ion Accumulation Time on the Detectability of Analytes in IR-MALDESI MSI.</a> Anal. Chem. 87, 10483-10490 (2015).</li><li>Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProteoWizard:+open+source+software+for+rapid+proteomics+tools+development.">ProteoWizard: open source software for rapid proteomics tools development.</a> Bioinformatics. 24 (21), 2534-2536 (2008).</li><li>Schramm, 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=ImzML+-+A+common+data+format+for+the+flexible+exchange+and+processing+of+mass+spectrometry+imaging+data.">ImzML - A common data format for the flexible exchange and processing of mass spectrometry imaging data.</a> J. Proteomics. 75 (16), 5106-5110 (2012).</li><li>Race, A. M., Styles, I. B., Bunch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusive+sharing+of+mass+spectrometry+imaging+data+requires+a+converter+for+all.">Inclusive sharing of mass spectrometry imaging data requires a converter for all.</a> J. Proteomics. 75 (16), 5111-5112 (2012).</li><li>Robichaud, G., Garrard, K. P., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSiReader:+an+open-source+interface+to+view+and+analyze+high+resolving+power+MS+imaging+files+on+Matlab+platform.">MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform.</a> J. Am. Soc. Mass Spectrom. 24 (5), 718-721 (2013).</li><li>Smith, C. A., O'Maille, G., 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=METLIN:+a+metabolite+mass+spectral+database.">METLIN: a metabolite mass spectral database.</a> Ther. Drug. Monit. 27 (6), 747-751 (2005).</li><li>Sud, M., 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=LMSD:+LIPID+MAPS+structure+database.">LMSD: LIPID MAPS structure database.</a> Nucleic Acids Res. 35, D527-D532 (2007).</li><li>Schwartz, S. A., Reyzer, M. L., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tissue+analysis+using+matrix-assisted+laser+desorption/ionization+mass+spectrometry:+practical+aspects+of+sample+preparation.">Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation.</a> J. Mass Spectrom. 38 (7), 699-708 (2003).</li><li>Takai, N., Tanaka, Y., Inazawa, K., Saji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+pharmaceutical+drug+distribution+in+multiple+organs+by+imaging+mass+spectrometry.">Quantitative analysis of pharmaceutical drug distribution in multiple organs by imaging mass spectrometry.</a> Rapid Commun. Mass Spectrom. 26 (13), 1549-1556 (2012).</li><li>Liu, J., Gingras, J., Ganley, K. P., Vismeh, R., Teffera, Y., Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+tissue+distribution+study+of+drugs+in+neonate+mice+using+desorption+electrospray+ionization+mass+spectrometry+imaging.">Whole-body tissue distribution study of drugs in neonate mice using desorption electrospray ionization mass spectrometry imaging.</a> Rapid Commun. Mass Spectrom. 28 (2), 185-190 (2014).</li></ol>

The authors declare no competing financial interests.

上述协议描述为执行IR-MALDESI MSI实验的关键步骤。矩阵应用程序（第3节）大约需要20分钟，这是通过升华或类似的用于MALDI微星实验的典型矩阵应用过程使用机器人喷雾器喷涂。此外，红外MALDESI不依赖于分析物的分配到基质晶体6和冰基质可普遍用于所有分析物，无论它们的质量，大小，或化学性质的。此外，利用冰的消除了在典型的MALDI实验23观察到较低的m / z范围的矩阵相关的离子。 虽然在实验中的所有步骤都需要产生质量MSI数据，在实验的过程中，电稳定性是可重复的IR-MALDESI离子发生是至关重要的。相对湿度需要在成像实验的过程中要监视保持恒定的冰基质层。对IR-MALDESI解吸是相对不透冰6的厚度;然而，一些成像实验可以采取多个小时，如果不能监控的冰层可能成为麻烦。为此，一个比例 - 积分 - 导数（PID）控制器最近被集成到源，以便监视机箱内的温度和相对湿度，并根据需要进行调整。 因为微星涉及从表面分析物的直接取样，色谱分离不能用来减小谱的复杂性。此外，目前在实验室环境中的环境的离子可以与来自目的分析物信号干扰。从环境离子光谱与复杂多样化的生物样品内的化合物的组合需要IR-MALDESI（和其他环境MSI源）以质量高精确度和高分辨率质谱仪耦合，SUCH在该实验中使用的，为了方便代准确和详细的离子地图之一。 在这项工作中，使用的IR-MALDESI揭示了多种在整个小鼠的各个器官内源性代谢物的空间分布。高峰在MSiReader采摘算法能够识别超过500个组织特异性离子，其中许多人来自不同的类脂质。该协议也可以用于同时监测外源化合物，例如药物和药物代谢产物的分布，在不同的器官24,25。黄金标准全身药物分布研究是全身放射自显影24。然而，该方法是耗时的，需要放射性标记的化合物和母体药物及其代谢物不能区分，因为它不提供有关分析物的分子结构的信息。全身微星与IR MALDESI规避这些问题，同时也使在周围环境中执行的分析，不加入有机基质。

在微探针模式质谱成像（MSI）通过在样品的表面上的离散位置的光束（激光或离子）涉及从表面样品的解吸。在每个栅格点，则产生的质谱和所获取的光谱，与从它们被收集的空间位置一起，可以用来同时映射样本内的许多分析物。此无标记耦合到灵敏度和质谱特异性成像的方式帮助微星成为质谱1,2-最快速发展的领域之一。 基质辅助激光解吸/电离（MALDI）是用于MSI分析中最常见的电离方法。然而，需要一种有机基质和MALDI的真空要求姿势上再现性，样品通量，并且可以使用该方法分析的样品的类型显著局限性。许多大气压力（AP）的IOnization方法已被开发，近年来，以规避这些限制3。这些环境电离方法允许生物样品的分析中是更接近自然状态，并简化分析前样品制备步骤的环境。基质辅助激光解吸电喷雾电离（MALDESI）是这样一种电离法4,5的一个例子。 在IR-MALDESI实验中，冰的薄层沉积在组织表面作为能量吸收基质上。中红外激光脉冲由冰基质吸收，并促进中性材料的解吸从由共振激励OH表面伸展的水模式。解吸的中立分区为正交电的带电滴，并且后电离在ESI状方式4-6。外源性冰矩阵的加法优于在组织上的内源性水单靠，因为它有助于交流算用于在不同组织区室含水量的变化，并且已显示通过〜15倍的组织成像实验7,8，以提高解吸6和提高离子丰度。 在这项工作中，我们采用了IR-MALDESI MSI引发的新生小鼠全身不同器官的代谢产物的分布。对IR-MALDESI源的可调节的参数的概况给出，并且对组织切片的成功的成像必要的步骤被证明。

<table><tbody><tr><td>IR-MALDESI Source</td><td>Custom-made</td><td>N/A</td><td>Please refer to references 4 and 12 for an in-depth discussion of IR-MALDESI source development.</td></tr><tr><td>Q Exactive Plus&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td></tr><tr><td>Water, HPLC Grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH365-4</td><td></td></tr><tr><td>Methanol, HPLC Grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH230-4</td><td></td></tr><tr><td>Formic Acid</td><td>Sigma Aldrich&amp;nbsp;</td><td>56302</td><td></td></tr><tr><td>Tunable mid-IR Laser</td><td>Opotek Inc.</td><td>IR Opolette</td><td>Tunable 2,700-3,100&amp;nbsp;nm IR OPO laser</td></tr><tr><td>Nitrogen Gas</td><td>Arc3 Gases</td><td>AG S-NI300-5.0</td><td>Grade 5.0 high purity nitrogen gas cylinder (300)</td></tr><tr><td>Cryostat</td><td>Leica Biosystems</td><td>CM 1950</td><td>Cryomicrotome</td></tr><tr><td>High Profile Microtome Blades</td><td>Leica Biosystems</td><td>3802123</td><td>Leica DB80HS</td></tr><tr><td>Mounting Medium (OCT)</td><td>Leica Biosystems</td><td>3801480</td><td>Surgipath FSC 22 mounting medium</td></tr><tr><td>Cryostat Specimen Disc</td><td>Leica Biosystems</td><td>14047740045</td><td>40 mm diameter</td></tr><tr><td>Glass Microscope Slides</td><td>VWR</td><td>48312-003</td><td>Frosted, selected, pre-cleaned</td></tr></tbody></table>

whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (ir-maldesi)

对于质谱（MS）的环境电离源已在过去的十年中很大兴趣的主题。基质辅助激光解吸电喷雾电离（MALDESI）是这样的方法，一个例子，其中基质辅助激光解吸/电离（MALDI）（ 例如，解吸的脉冲性质）和电喷雾电离（ESI）（ 例如，软电离的特征）组合。之一的MALDESI的主要优点是其固有的多功能性。在MALDESI实验中，紫外线（UV）或红外线（IR）激光可用于共振激发内源或外源矩阵。矩阵的选择不是分析物依赖性的，并且仅取决于用于激发的激光波长。在IR-MALDESI实验中，冰的薄层沉积在样品表面作为能量吸收基质上。对IR-MALDESI源几何已经使用的试验设计（DOE），用于液体样品的分析统计设计以及生物化学优化ogical组织标本。此外，一强大的红外MALDESI成像源已被开发出来，其中，可调谐的中红外激光器用计算机控制的XY平移阶段和高的分辨能力质谱仪同步。自定义的图形用户界面（GUI）允许激光的重复频率的用户选择，每像素镜头，样品台的步长，和解吸之间的延迟和扫描源的事件的数目。红外MALDESI已在多种应用中被使用，如生物组织切片的纤维和染料和MSI的取证分析。不同的分析物，从内源性代谢物的组织切片中的外源外源物的分布可以被测量，并使用这种技术进行定量。在这个手稿介绍的协议详细描述了全身组织切片的IR-MALDESI MSI主要步骤。

在图4中呈现的图像显示在不同器官中的全身组织切片的代谢物的空间分布。使用MSiReader PeakFinder发现独特m / z值到身体的特定区域，接着通过分批处理的图像生成。图像叠加工具（ 图3-4）被用来对准所得离子地图冰基质沉积之前拍摄的光学图像。胆固醇是在所有组织类型观察到如从动物细胞膜（ 图4A）其结构的作用预期的，而其他的代谢物显示出特定的器官或组织隔室（ 图4B-D）的范围内不同的分布。 <img alt="图4" src="/files/ftp_upload/53942/53942fig4.jpg" /> 图4.代表性的IR-MALDESI MSI图像; AD四个 Metaboli游戏TE离子的地图展现着整个鼠标部分的化合物的本地化。脂质类在每幅图像的右下角注释。组织切片的光学图像是为了与器官的可视化，以帮助叠加离子的地图范围内。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53942/53942fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI at JoVE.com

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 ...

whole-body-mass-spectrometry-imaging-infrared-matrix-assisted-laser

Nanyang Technological University

Research

JoVE Journal

Bioengineering

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

9.4K 次观看.  North Carolina State University. 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. 

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

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

基质辅助飞行的完整蛋白质（MALDI-TOF）质谱分析激光解吸/电离飞行时间大于100 kDa的

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

科研

化学

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 ...

生物化学

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 ...

生物工程

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

利用成像质谱技术捕获组织与细胞之间的小分子通信

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

癌症研究

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

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

神经科学

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 ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

MALDI-质谱成像代谢物的研究<em&gt;苜蓿</em&gt;根瘤

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

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

Introduction to Mass Spectrometry

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.
A mass spectrometer is composed of an ionization source, an analyzer,&#160;and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.
In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.