We would like to acknowledge the technical assistance of Kristina Jurcic in the University of Western Ontario MALDI MS facility, as well as the National Sciences and Engineering Council (NSERC) for funding this work. The authors would also like to acknowledge the Caprioli group (Vanderbilt University, TN) and Chaurand group (Universit&#233; de Montreal, QC) for their advice in optimizing the sublimation technique presented in this manuscript.

坚持以下描述西方安大略省的动物护理委员会（2016-014）的大学所有的动物处理程序。 1.组织准备和剖分<ol><li>通过所谓的"新鲜冷冻萃取"（FFE）的过程中提取从大鼠脑组织。 <ol><li>安乐死大鼠的戊巴比妥钠的过量和监测四肢反射。一旦所有的反射都停止了，断绝使用断头台鼠的头上。 </li><li>仔细的肌肉和其他组织使用手术刀颅骨分离。使用骨切割钳打破颅骨以暴露脑，从头骨（枕骨大孔）的底部开始到前部。 </li><li>一旦大脑暴露出来，小心地舀出来用手术锅铲，立即放在闪存冷冻碎干冰。 注:大脑会很可塑性。因此，放置在大脑时要格外小心干冰。如果大脑被压碎或针对一个表面按压时，它会改变其形状和在该位置结冰。 </li></ol></li><li>取下新鲜冷冻组织（未用甲醛灌注或嵌入在OCT） - 80℃的冰箱中放干冰。 </li><li>放置几滴水上低温恒温器保持器和地点上或干冰或低温恒温器冻结栏。按组织上轻轻低温恒温器支架，因为它开始冻结并按住，直到组织的基地周围水结冰和主持人到位。添加额外的水，如果必要，以进一步固定在支架上的组织。 注:水是用来代替OCT介质中，以避免与这会影响所希望的信号的检测化合物的组织的污染安装组织。 </li><li>位置组织低温恒温器。部组织到所需的解剖位置。确保切割厚度为8之间 - 12微米。 注:当切片在公共组织的低温恒温器设备，当务之急是独立的材料，如刀片，刷子，防倾杆，并持有人按顺序使用，以避免与嵌入化合物污染。低温恒温器的内也应在使用前与乙醇彻底清洁。 </li><li>使用低温恒温器防倾杆，慢慢的部分扁平的组织切片。如果倾杆的位置不正确，或刀片变钝可以发生在组织孔或标记。移动至组织，使用干净的漆刷切片平台的中心。 </li><li>安装<ol><li>通过将它们放置在低温恒温器，而切片冷冻导电幻灯片或金属板。当滑动被完全冻结，小心移动切片组织上使用清洁漆刷滑动的导电表面。一旦所有的组织切片的幻灯片正确定位，请将手指滑动下，组织对面，按直到部分融化。 注:章节可以折叠或在解冻过程中，当卷曲使用这种安装方法。这可以通过解冻组织时保持滑动在低温恒温器被减少。如果这些问题成为问题，或者，预热安装方法如下。 </li><li>可替代地，采取室温铟锡氧化物（ITO）滑动（或金属板），并轻轻地向下压上的切片平台表面冷冻组织部分，导电性的一面朝下。这将导致该组织部分解冻均匀到具有小卷曲或组织的折叠滑动的表面上。 注意:使用这种方法可能导致某些蛋白质和脂质的损失时安装冷凝可能会出现部分的下方。 </li></ol></li><li>放置与组织切片中进行5干燥器 - 10分钟。 </li></ol> 2.升华器设备建立注:在通风橱中执行这些步骤。 <ol><li>放置在铝容器中的砂浴上热板，以在金属剪式升降热板适当的表面积（ 即比热板大）。开启热板，并设置温度至140℃。 注:熔点DAN矩阵是187之间 - Visual C 190°。不要超过在烤盘这个温度。 <ol><li>如果加热板配有一个温度反馈探针，用它来监视砂温度在整个实验，确保温度一致。此功能可以帮助实验在不同的升华实验重复性。 注:砂浴应铝容器如玻璃容器内被包含在高温下可能碎裂。 </li></ol></li><li>放置300毫克DAN基质上升华装置的底表面上。放置基质在该装置的中心，并在近似宽度和滑动的长度的偶数层摊开被升华。 注意:DAN矩阵是有毒的。因此，重要的是戴手套，口罩，和安全在任何时候都护目镜处理粉状基质时。 DAN应贮存在黑暗中，因为它是光敏感。 </li><li>带的金属板到具有制版与冷凝器的底部直接接触，以确保均匀的温度的升华期间横跨滑板的整个表面的冷却分布装置的内表面上。可替代地，砂冷凝器的底部，以确保一个平的表面。 <ol><li>放置胶带沿板的外边缘和附着到内玻璃器皿的侧面。如果磁带被放置在板下，并附着在内层玻璃表面的底部，温度分布可能不连，并可能导致在横跨滑板的表面凹凸不平矩阵分布。 </li><li>带的空白（测试）跨越与再次放置在滑动件的外边缘带的金属板的表面上对角滑动。 </li></ol></li><li>连接装置的顶部和底部，W第i个中间橡胶O型圈，以确保完全密封。 </li><li>放置一个金属U型关节周围的装置的中心并拧紧虎钳直到该装置的顶部和底部一半被密封紧密在一起。在金属O型圈的沙浴以上的地方。 </li><li>以碎冰一把，并将其放置在冷凝器中。填写冷凝器¼到½充分用冷水创建冰凌。冰雪泥会冷却在装置的内部，并且随后将滑动附着于它的金属板。等待至少5分钟，使温度达到稳定状态。 </li><li>倒300毫升乙醇中的冷阱容器和玻璃器皿放置到容器中。降2 - 3小块干冰入乙醇中的冷阱容器的底部。冷阱输入具有运行在气缸底部的玻璃管，而不会输出。 </li><li>连接真空泵使用橡胶管冷阱输出。用另一块橡胶油管冷阱输入连接到升华设备。确保油管（如果可用）使用金属夹子关紧。 </li><li>使用真空泵以提供30的真空 - 为50mT。允许泵为至少5分钟，使压力平衡运行。升华装置的U形环上的虎钳可能必须被重新拧紧，一旦真空泵被接通，因为在该装置中的压力降低上。 注:强烈建议真空计被连接到泵以监测在实验过程中的真空的压力和以达到实验之间的最高可能的再现性来测试泄漏的压力。然而，大多数的泵被设计成维持恒定的压力，因此该计可能不是经验丰富的用户是必不可少的。此外，真空密封润滑脂可用于帮助维持真空压力。 </li></ol> 3.升华<ol><li>确保砂浴温度具有STABIL源化在140℃，并且该装置被固定在该金属O型环的砂浴具有连接所有管路的上方和真空接通。 </li><li>设置计时器7分钟，但不启动定时器。慢慢地将剪叉式升降机和沙浴直至升华设备，直至升华的万向节是很好的金属O型圈以上。这额外的空间允许在砂设备的调整。 <ol><li>迅速轻轻按下升华的砂表面上的，以确保该装置是坐在均匀在沙滩上，然后立即启动定时器。 </li></ol></li><li>当计时器的声音，关闭真空泵，并小心地将剪叉式升降机，直到升华不再接触沙浴，并在金属O型圈安全坐着。 <ol><li>缓慢地松开微排气阀来释放在装置的压力。松开周围的升华设备的橡皮管金属夹，慢慢地开始放松管。一旦RUBBER管已略有松动，弯曲管一侧，使残余压力逃脱。 </li><li>当环境压力的回报，小心地取出从升华设备的橡胶管。 </li></ol></li><li>松开装置的万向节的恶习，并删除。仔细分离升华装置的两个半部，并从内玻璃器皿的顶部拉断的幻灯片。检查幻灯片确认连矩阵分布。 注意:如果矩阵是不均匀的，在升华的底部粉状基质可以重新定位或升华器设备所用的砂为下一幻灯片重新定位。升华工序可以重复使用相同的矩阵数次，但是，基质最终将成为反复热暴露和粉末的量更暗将减小，使得升华时间将必须稍微调整以补偿。出于这个原因，重要的是监测矩阵贝的量是非常重要的纳克实验后升华，以确保幻灯片之间的基质沉积的一致性</li><li>如果基质的升华的分布和数量是足够的，用胶带与组织新的幻灯片到升华器，并重复第3节的内侧。 注:对于质量控制（QC）的目的，升华基质的量可以在每个实验后通过称量滑动之前和升华后除以与滑动2的表面积升华基质的重量计算，其应注意的是由于缺乏超过4小数点和鳞之间的变异最秤的精度，这些测量应仅用于最佳基质沉积的引导，而不是质量控制的一个完全量化手段，除非使用高精密天平（ 见图3）。 </li></ol> 4.组织存储/补液<ol><li>升华后，储存滑动在密封容器或小卡斯ETTE在密封的塑料袋中。 </li><li>孵育在-20℃冷冻载玻片2小时或过夜。 注:冷冻过程的目标是充当组织材料的解吸到矩阵的复水的步骤。冷冻过程也被证明当储存在-80℃下12以防止脂质的信号的劣化长达1周。出于这个原因，时间的样值存储在冰箱的量可以改变，以一定的程度，以满足实验者的需要而不显著改变的信号检测（如在我们的实验室观察到的）。但是，我们已经注意到矩阵的一些变色当样品在-20℃冷冻的时间超过24小时。因此，我们建议成像升华组织该时间之前或在-80℃储存组织更长的潜伏期。 </li><li> 10分钟 - 在5干燥器卸下从冰箱（集装箱），地点幻灯片。 </li></ol> 5.成像和Analysis <ol><li>从干燥器中取出的幻灯片和应用仪器标准，以确保在成像MALDI仪器的质量精确度。的类型的标准将取决于仪器而有所不同。标准一般横跨整个幻灯片施加均匀，周围组织将被成像（ 图3D）。 </li><li>将滑入MALDI仪器，并按照制造商的成像实验说明（仪器方法应针对1000进行优化 - 2000质量范围）。代表性的结果（ 图4）在反射器负模式获得具有70微米的光栅和20张/谱（采集时间〜2小时）。 </li></ol>

<ol>
	<li>Hankin, J. A., Barkley, R. M., Murphy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublimation+as+a+method+of+matrix+application+for+mass+spectrometric+imaging.">Sublimation as a method of matrix application for mass spectrometric imaging.</a> J. Am. Soc. Mass Spectrom. 18, 1646-1652 (2007).</li><li>Thomas, A., Charbonneau, J. L., Fournaise, E., Chaurand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublimation+of+new+matrix+candidates+for+high+spatial+resolution+imaging+mass+spectrometry+of+lipids:+enhanced+information+in+both+positive+and+negative+polarities+after+1,5-diaminonapthalene+deposition.">Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition.</a> Anal. Chem. 84, 2048-2054 (2012).</li><li>Caughlin, S., 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=Increased+Expression+of+Simple+Ganglioside+Species+GM2+and+GM3+Detected+by+MALDI+Imaging+Mass+Spectrometry+in+a+Combined+Rat+Model+of+A&#946;+Toxicity+and+Stroke.">Increased Expression of Simple Ganglioside Species GM2 and GM3 Detected by MALDI Imaging Mass Spectrometry in a Combined Rat Model of A&#946; Toxicity and Stroke.</a> PLoS ONE. 10, 0130364 (2015).</li><li>Weishaupt, N., Caughlin, S., Yeung, K., Whitehead, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Anatomical+Expression+of+Ganglioside+GM1+Species+Containing+d18:1+or+d20:1+Sphingosine+Detected+by+MALDI+Imaging+Mass+Spectrometry+in+Mature+Rat+Brain.">Differential Anatomical Expression of Ganglioside GM1 Species Containing d18:1 or d20:1 Sphingosine Detected by MALDI Imaging Mass Spectrometry in Mature Rat Brain.</a> Front Neuroanatomy. 9 (155), (2015).</li><li>Whitehead, S., 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=Imaging+Mass+Spectrometry+Detection+of+Gangliosides+Species+in+the+Mouse+Brain+following+Transient+Focal+Cerebral+Ischemia+and+Long-Term+Recovery.">Imaging Mass Spectrometry Detection of Gangliosides Species in the Mouse Brain following Transient Focal Cerebral Ischemia and Long-Term Recovery.</a> PLoS ONE. 6 (6), 20808 (2011).</li><li>Fuchs, B., S&#252;&#223;, R., Schiller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+of+MALDI-TOF+mass+spectrometry+in+lipid+research.">An update of MALDI-TOF mass spectrometry in lipid research.</a> Progress in Lipid Research. 49, 450-475 (2010).</li><li>Barcel&#243;-Coblijn, G., Fern&#225;ndez, J. 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+spectrometry+coupled+to+imaging+techniques:+the+better+the+view+the+greater+the+challenge.">Mass spectrometry coupled to imaging techniques: the better the view the greater the challenge.</a> Front Physiol. 6 (3), 1-5 (2015).</li><li>Angel, P. M., Spraggins, J. M., Baldwin, H. S., Caprioli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sensitivity+for+high+spatial+resolution+lipid+analysis+by+negative+ion+mode+matrix+assisted+laser+desorption+ionization+imaging+mass+spectrometry.">Enhanced sensitivity for high spatial resolution lipid analysis by negative ion mode matrix assisted laser desorption ionization imaging mass spectrometry.</a> Anal. Chem. 84, 1557-1564 (2012).</li><li>Murphy, R. C., Hankin, J. A., Barkley, R. M., Zemski Berry, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+imaging+of+lipids+after+matrix+sublimation/deposition.">MALDI imaging of lipids after matrix sublimation/deposition.</a> Biochim. Biophys. Acta. 1811, 970-975 (2011).</li><li>Jaskolla, T. W., Karas, M., Roth, U., Steinert, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+vacuum+sublimed+matrices+and+conventional+dried+droplet+preparation+in+MALDI-TOF+mass+spectrometry.">Comparison between vacuum sublimed matrices and conventional dried droplet preparation in MALDI-TOF mass spectrometry.</a> J. Am. Soc. Mass Spectrom. 20, 1104-1115 (2009).</li><li>O'Rourke, M. B., Raymond, B. B., Djordjevic, S. P., Padula, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+cost-effective+method+for+the+analysis+of+fresh+frozen+tissue+sections+via+matrix-assisted+laser+desorption/ionisation+imaging+mass+spectrometry.">A versatile cost-effective method for the analysis of fresh frozen tissue sections via matrix-assisted laser desorption/ionisation imaging mass spectrometry.</a> Rapid Commun. Mass Spectrom. 29, 637-644 (2015).</li><li>Patterson, N. H., Thomas, A., Chaurand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+time-dependent+degradation+of+phospholipids+in+sectioned+tissues+by+MALDI+imaging+mass+spectrometry.">Monitoring time-dependent degradation of phospholipids in sectioned tissues by MALDI imaging mass spectrometry.</a> J Mass Spectrom. 49, 622-627 (2014).</li><li>Cheng, H., Sun, G., Yang, K., Gross, R. W., Han, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+desorption/ionization+of+sulfatides+by+MALDI-MS+facilitated+using+9-aminoacridine+as+matrix.">Selective desorption/ionization of sulfatides by MALDI-MS facilitated using 9-aminoacridine as matrix.</a> J. Lipid Res. 51, 1599-1609 (2010).</li><li>Puolitaival, S. M., Burnum, K. E., Cornett, D. S., 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=Solvent-free+matrix+dry-coating+for+MALDI+imaging+of+phospholipids.">Solvent-free matrix dry-coating for MALDI imaging of phospholipids.</a> J. Am. Soc. Mass Spectrom. 19, 882-886 (2008).</li><li>Chaurand, P., Cornett, D., Angel, P., Caprioli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Whole-body+Sections+Down+to+Cellular+Level,+Multiscale+Imaging+of+Phospholipids+by+MALDI+Mass+Spectrometry.">From Whole-body Sections Down to Cellular Level, Multiscale Imaging of Phospholipids by MALDI Mass Spectrometry.</a> Mol Cell Proteomics. 10, (2011).</li><li>Grove, K. J., Frappier, S. 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=Matrix+pre-coated+MALDI+MS+targets+for+small+molecule+imaging+in+tissues.">Matrix pre-coated MALDI MS targets for small molecule imaging in tissues.</a> J. Am. Soc. Mass Spectrom. 22, 192-195 (2011).</li><li>Yang, J., 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=Matrix+precoated+targets+for+direct+lipid+analysis+and+imaging+of+tissue.">Matrix precoated targets for direct lipid analysis and imaging of tissue.</a> Anal. Chem. 85, 2907-2912 (2013).</li><li>Gemperline, E., Rawson, S., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+comparison+of+multiple+MALDI+matrix+application+methods+for+small+molecule+mass+spectrometric+imaging.">Optimization and comparison of multiple MALDI matrix application methods for small molecule mass spectrometric imaging.</a> Anal. Chem. 86, 10030-10035 (2014).</li></ol>

The authors have nothing to disclose.

这项工作细节矩阵升华对组织的标准化协议，用于检测带负电荷的脂类，如神经节苷脂，在MALDI IMS实验。用于MALDI IMS的样品制备是高度可变的，并且必须被定制，以适应所关注分析物的独特性能。矩阵的上组织样品表面上的应用程序是在样品制备过程的与问候结果在IMS中的质量的一个关键方面。应特别注意选择矩阵时，确保该矩阵是与所关注的分析物相容并且是自由的频谱矩阵衍生工件作出。升华干燥的基质沉积技术，它提供了对组织脂类的小离域的高度矩阵液晶同质化。特别DAN矩阵示出了用于一组带负电荷的膜脂质称为神经节苷脂的解吸高度敏感性的，这表现在大鼠脑部分，并且是真空延长的时间周期下是稳定的，允许与大多数成像应用的兼容性。 DAN的矩阵中有还具有用于带正电荷的脂质种类的亲和性高，因此增加了该矩阵用于MALDI IMS应用2的多功能性的额外好处。还应当指出的是，多个脂质物种，包括磷脂，可以同时使用该IMS协议时检测到。 相比9等常用基质DAN矩阵的性质进行彻底检查，已经出版2。在这篇文章中，相对于检查以及能力使用DAN高分辨率成像其他矩阵的作者强调DAN矩阵的负离子模式的敏感性增加。 DAN矩阵被发现具有负极性的离子的最高的总体性能。有趣的是，它执行EQUALLY以及为正极性离子。已证明高亲和性为负极性离子其他常见的基质包括DHA，DHB，和9-AA。 DHA的作为基体的效率是通过在真空下其稳定性低，这使得它不切实际的大多数成像应用2的限制。 9-AA具有产生低的背景噪声，并在750硫脂信号表明富集的好处-相比DHB基质13在负模式950质量范围。虽然DHB已经显示出在正模式非常有效，它产生较低的负极性离子信号相比DAN基质2作为。 DAN基质也证明在较低质量范围（650 - 950）在负离子模式增加灵敏度相比DHB 2。此外，已经报道，DHB可以形成在负离子模式下高达750达2显著矩阵簇。 等人描述。 ，其中地面基质通过一个细筛并沉积到样品表面。作者比较了基质沉积到湿基手工喷涂技术，这种干法，发现他们给了相同的信号在450磷脂- 1200质量范围14。一项研究同时使用升华和干燥的涂料用筛子检查磷脂的表达，但是，作者并没有在这两种技术在图像分辨率或离子信号产量15方面相比如何评论。 为了保证适用性最广泛的，介绍的协议是一项基本的工作流程，使MALDI IMS的新的和更专业的用户实现高度可重复的结果各种各样的脂质成像应用。然而，升华的协议可以被修改，以满足uniqu实验电子的情况。例如，通过升华提供的细矩阵晶体沉积允许扭转由预涂布载玻片样品制备的顺序/事先安装所述组织与矩阵板的选项。矩阵可以预先升华到所述导电表面，并存储在供以后使用从而减少样品制备时间切片后，同时仍然保持对脂质检测16,17高灵敏度的黑暗的环境中。可以作出此协议，以提高信号的检测脂质的另一个修饰是2分钟洗涤用甲酸铵8。 虽然升华可以规避许多最湿沉积技术，如较小的缺点，更均匀的基质晶体沉积1相比，自动喷雾器分析物的离域，而且成本低降低，所以我在样品制备过程的变化是不可避免的。在升华的情况下，有几个因素可能会导致从一个实验到另一分钟的差异，并且可以包括已在我们的实验室中观察到以下内容。可以存在于上砂的升华装置的位置的差异。当运行多个升华实验后端到背面，在沙浴中的砂可能会开始改变其位置，这可能会影响整个砂浴的热量的分散。每一轮升华之前压扁的沙子也可以改变热量的分散为已流离失所可能需要一段时间才能恢复到均匀的温度沙子。升华时间可能要每一轮之间小幅调整，以补偿在沙浴流沙。可替代地，使用油浴的可能导致更均匀的热分散。 其次，基质中的升华的底部的量可以变化。 DAN马特里x的块状灰色的外观，并具有趋向于形成大簇。这些集群可以手动打破并布置在升华的底部，但不能完全消除。矩阵的大簇需要更长的时间来加热和升华比小块可影响升华的最终产品。 在升华装置中的真空输出是另一个重要的因素。大多数sublimators与单个真空输出到其管道连接升华期间将其连接到真空泵制成。因为压力被在装置的一侧调节，可能存在的基质沉积的轻微不均匀，与更多的沉积在与真空输出侧。这可以通过改变在沙装置的任一位置，在升华的底部基质的位置，或通过改变冷凝器上的滑动/板的位置有所补偿。这些事实ORS是升华技术的主要缺点，为此它是至关重要的运行试验样品，以便时间可以调整，以弥补这些变量之前运行通过升华工序的测试幻灯片。 升华的另一报告缺点是更高质量的分析物的检测是通过分析物的提取不充分和/或与基质混合的限制。升华后，再水合步骤可以帮助拉出这些更高质量分析来规避这个问题。这是通过使用一湿度室18典型地实现。然而，在该协议中，一个冷冻步骤是升华而达到相同的目的之后加入。样品的插入之前在MALDI仪器冷冻和其后的解冻（在干燥器）导致缩合，以形成样品的表面暂时再水合的样品，以允许更高质量的脂质的提取，同时保留在Ti上ssue用于分析12,17。 总体而言，升华是用于检测在MALDI IMS实验脂质的基质应用的一个高度敏感的和具有成本效益的方法。实际上，当我们从使用空气喷涂法3为基质沉积升华法4移位我们小组已经注意到对IMS结果显著改进。本协议是适当的数目的脂质成像应用，但可以进行修改，以适应实验者的需要。

基质辅助激光解吸/电离（MALDI）成像质谱（IMS）正在成为一种高度寻求技术为脂质，肽和蛋白质穿过完好样品表面的空间分布的可视化之后。 MALDI IMS的以前被称为预纯化的分析物的分析技术，但在最近几年中，已经在因为具有高分辨率视觉/解剖参考点，而质谱的精确度结合的能力许多其他学科提请注意需要任何外部标记。作为利用这种技术的研究的科学池继续增长，所需要增加的标准化，易于后续的协议，以协助IMS实验开发和优化。神经节苷脂，一组膜脂质在中枢神经系统高度丰富的，是理想的MALDI IMS实验作为它们的位置，嵌入在膜中，使某些特定上课不可能使用常规的免疫标记以检测。此外，我们已经表明，使用MALDI IMS中，这些脂质，其充当细胞信号的调节剂，除其他事项外，有在被脑损伤3，4，5后改变了健康啮齿动物脑独特的解剖分布模式。神经节苷脂位于较高质量范围相比，大多数脂质种类，并因此最适合于MALDI成像平台。 <img alt="图1" src="/files/ftp_upload/55254/55254fig1.jpg" /> 图1:MALDI IMS实验的工作流程。使用升华在MALDI IMS实验的一般流程的图。组织在-80℃冷冻于低温恒温器被分段和10微米的部分是安装在导电的ITO载玻片解冻。然后将载玻片置于干燥器直到升华。 SLIDES插入升华装置和偶数层矩阵施加到组织样本表面。样品在-20℃冷冻然后置于干燥器中10分钟，冷冻过夜。一旦标准被应用，将样品插入到其中激光跨越引起解吸分子在基质中以电离所述组织指示MALDI仪器。离子行进向下根据它们的质量（时间飞行/ TOF）直到它们到达了检测器的飞行管和分离。上一个预定的质量 - 电荷（M / Z）范围内的离子丰度分析物的信息显示为两个分子图像和质谱。这个数据可以用来既可视化和量化的成像组织内感兴趣的分析物的离子丰度。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55254/55254fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 样品制备用于MALDI IMS是高度可变的过程的每个步骤必须进行定制，以所关注的分析物。基于MALDI-实验限定特征是使用沉积到分析前的样品表面上的基质的涂层。除了吸收和在消融过程中从激光转印辐射能量的作用，该基质也用于各种分析物从样品中分离，从而有利于感兴趣6,7化合物的分析。基质样品表面的均匀的应用是在样品制备过程中最关键的步骤。不正确的基质沉积可导致大量异构基质晶体形态和文物的发展，低离子信号，重复性差7。 由于某些矩阵的亲和力来分离特定的分析物，被选择用于实验矩阵的类型可以显著改变的结果。用于蛋白质和肽的成像矩阵常常不同于用于成像脂质不同，此过程是通过需要额外的方法，如为了从组织成功地检测信号的洗涤和再水合步骤进一步复杂化。虽然脂质信号8的增强存在洗涤步骤，它们不是用于检测最脂质种类的一个先决条件。当选择的脂成像实验的矩阵，它考虑的兴趣，因为这脂质的极性将缩小合适矩阵的范围是重要的。例如，神经节苷脂含有唾液酸残基这给他们一个整体的负极性。有许多的矩阵，可以有效地解吸和电离来自组织神经节苷脂;然而，因素，如在频谱并在真空下基质的稳定性基质衍生的峰必须在考虑服用。 1,5-diaminonapthalene（DAN）矩阵是仪器的真空条件对于大多数成像应用下足够稳定，并表现出高度的脂质解吸灵敏度和可用于在正和负离子模式2脂质的分析。 DAN基质，相对于其他负脂质亲和基质如二羟基苯甲酸（DHB），9-氨基吖啶（9-AA）和5-氯-2-巯基苯并（CMBT）时，能够最有效地解吸从大鼠脑神经节苷脂组织在负离子模式（手稿准备中）。 选择基质沉积的适当的方法是同等重要选择矩阵本身。湿基质沉积方法，其中所述固体基质溶解在有机溶剂中，并通过气动沉积或自动喷雾器或检举，作为液体渗透的样品，以允许额外的用于蛋白质和肽的解吸特别有效化合物和共结晶与基质的ction。尽管这些技术也可用于脂质应用，分析物离域和不均匀的基质晶体地层是屡见不鲜由于脂质的在溶剂中的高丰度和溶解度，特别是在组织2,9。因为脂质易于从组织电离，干燥基质沉积技术，如升华，提供一种简单的，成本效益的替代喷雾器而绕过许多这些技术的缺点的。升华的MALDI IMS实验的成功归功于功能，如微晶矩阵的形态这增加了矩阵分析物结合的表面面积，增加基质纯度和均匀的基质的沉积，从而增加可重复性比较潮湿的矩阵技术1，10。 升华invoLVES正下方产生的固体，粉状基质随后沉积在组织样品表面的气相过渡冷却的样品表面加热真空下粉状基质。期间升华，基质沉积可通过改变因素例如时间，温度和压力，以提供高度可重复的结果来控制。单个升华试验可以在任何地方需要5至20分钟取决于矩阵的选择的类型，处理前可重复使用数次。该装置可商购于自动喷雾器的价格的几分之一处购买，并且很容易被拆开进行清洁和维护。成本低，这种基质沉积技术相对简单使其非常适用于开始后，或MALDI IMS脂质成像应用拓展的研究人员。虽然详细说明了组织对IMS的升华协议的信息已报告11，少数标准化协议存在W¯¯HICH重点涉及进行升华实验在负离子模式成像高质量脂质，使得难以建立的技术中没有广泛的试验和错误的基本工作流程。下面是一个试验性协议旨在填补DAN矩阵的升华到大鼠脑切片用于高分辨率成像和神经节苷脂的检测该间隙。 <img alt="图2" src="/files/ftp_upload/55254/55254fig2.jpg" /> 图2:升华设备。照片（A）和升华装置的示意图（ 二 ）。真空泵由橡胶管连接到冷阱充满300毫升乙醇。冷阱，然后由橡胶管的升华装置相连。该装置是由两个独立的玻璃器皿的件所用的金属U形接头密封在一起。升华器的上半部分包含填充有冰凌冷凝器。该样品板贴到所述冷凝器的底部，所述密封的玻璃装置内。升华设备的下半部分包含DAN矩阵，摊开均匀面临的样板。期间升华，玻璃装置被放置在砂浴中加热至140℃用热板正下方。温度探头有助于维持整个升华试验通过沙浴温度的反馈相比为实验的预设温度稳定的温度。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55254/55254fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Sublimator</td><td>Chemglass Life Sciences</td><td>CG3038-01</td><td></td></tr><tr><td>1,5-Diaminonapthalene (DAN) matrix</td><td>Sigma-Aldrich</td><td>D21200&amp;nbsp;</td><td>100 G</td></tr><tr><td>Cryostat</td><td>Thermo-Fisher Scientific</td><td></td><td>CryoStar NX50</td></tr><tr><td>Hot plate with temperature feedback</td><td>Thermo-Fisher Scientific</td><td>HP88857290</td><td>Isotemp ADVD 7x7 HP 100 - 120 V</td></tr><tr><td>Stainless Steel Jack</td><td>Thermo-Fisher Scientific</td><td>2216479</td><td>10x10</td></tr><tr><td>Cold Trap</td><td></td><td></td><td>Custom built on site</td></tr><tr><td>Vacuum Pump</td><td>Franklin Electric</td><td>1102180403</td><td>Savant VP100 Two Stage</td></tr><tr><td>Indium-tin-oxide (ITO) Slides</td><td>Hudson Surface Technology</td><td>PSI 1111000</td><td>type II, 1.1 mm/25 each</td></tr><tr><td>MALDI TOF/TOF 5800 Instrument</td><td>AB Sciex</td><td></td><td></td></tr><tr><td>Desiccator</td><td>Sigma-Aldrich</td><td>D2797</td><td>tabletop desiccator</td></tr></tbody></table>

sublimation of dan matrix for the detection and visualization of gangliosides in rat brain tissue for maldi imaging mass spectrometry

样品制备为最佳检测和基质辅助激光解吸/电离（MALDI）成像质谱（IMS）的实验分析物的可视键。确定适当的协议遵循整个试样制备过程中可能是困难的，因为每个步骤必须进行优化以符合与所关注分析物的独特的特点。这一过程不仅涉及发现能解吸和有效地电离感兴趣的分子相容的基质，而且选择合适的基质沉积技术。例如，一个湿基质沉积技术，这需要在溶剂中的基体溶解，优于大多数蛋白质和肽的解吸，而干燥基质沉积技术对于脂质的电离特别有效。升华已被报告为干燥基质沉积用于检测通过MALDI IMS在组织脂类的一种高效的方法，因为该homogenei相比于许多湿沉降方法1，2基质晶体沉积和最小的离域分析的TY。广泛地说，它包括将一个样品和粉状基质与按压冷的表面的采样的真空密封室中。然后该装置被降低到加热浴（砂或油），结果在粉状基质的升华到冷却组织样品表面。在这里，我们描述了使用1,5-二氨基萘（DAN）矩阵为使用MALDI IMS在大鼠脑神经节苷脂的检测和可视化升华协议。

经升华实验结束后，将玻璃仪器的两半在通风柜和滑动分离，贴在冷凝器，可以去除（ 图3A）。此时，滑动应检查在沙浴中的装置的不均匀矩阵分布和时间，温度，或位置可能必须调整为下一幻灯片。一个成功的DAN升华实验将导致甚至涂层沿着那里的组织的解剖特征可以清楚地显现在滑动和载玻片上基质的量的表面灰色/褐色矩阵的类似于量在组织上（ 图3B）。例如，如果过多基质已经升华在组织上，基质的厚度将覆盖组织和唯一的一般形状的特征将是可以辨认的。然而，如果太少矩阵升华，组织将哈已经比滑动（ 图3C）的其余部分更暗的外观。既过多和过少的矩阵将导致在MALDI仪器差信号。对于质量控制的目的，托马斯等人。称量附着在滑动基质的量和由滑动表面面积除以重量。他们报告基质沉积的最佳量为几个矩阵包括DAN（110微克/平方厘米）2。虽然大多数余额缺乏复制这样的结果所需的精度，我们试图QC这种方法;然而，由于高度的变异性，我们只能建议要关联发现基质沉积的一个合适的范围与成功与丹矩阵不成功的升华实验，通过仪器信号检测来确定。低于100微克/平方厘米的基质测定用"太少"矩阵的条件有关，而高于140微克/平方厘米的测量是与"太多"有关。 100和140微克/平方厘米之间基质沉积被认为是用于与DAN矩阵成像的最佳量。校准标准应在组织样本周围的载玻片被应用，以确保检测到的IMS信号的质量准确度（ 图3D） 在一个电离的IMS实验中，分子图像允许所关注的分析物的任何未知物种可能存在于一个给定的质量范围沿空间分布的可视化。神经节苷脂在该实验的分子图像使用ImageJ软件，以显示几种神经节苷脂物种横跨鼠脑（ 图4A）的皮质和皮质下区域的解剖分布重叠。在大脑中最丰富的神经节苷脂是A系列品种神经节苷脂GD1a和GM1而且还含有可以全部之间找到GM2神经节苷脂GM3和1000-2000 M / Z质量范围，更高的质量范围比最常见的脑脂质，使得这些神经节苷脂易于沿谱（ 图4B）确定。各神经节苷脂物种的离子丰度可用于半定量同一图像内神经节苷脂物种差异或变化。因为变异性的样品制备一定量的无法避免在IMS中，间扫描的比较被认为是半定量的。 <img alt="图3" src="/files/ftp_upload/55254/55254fig3.jpg" /> 图3. DAN升华。对大鼠脑组织DAN矩阵的升华的代表性结果。 （A）在升华后，该装置的两半分开，并在滑动从冷凝器除去。 （B）的DAN矩阵跨越多个大鼠脑组织切片均匀地分布在滑动的表面上，以允许高度再版oducible结果（100之间 - 150微克/平方厘米）。 （C）的，其中太少矩阵（左- &lt;90微克/平方厘米）或过多矩阵（右- &gt;失败升华实验实施例150微克/平方厘米）沉积。太少矩阵将导致分析物的解吸不充分，而太大矩阵将不允许激光采集期间穿透到组织，导致低离子检测。 （D）含有与施加DAN矩阵和校准标准升华大鼠脑组织切片幻灯片是准备用于插入到用于成像的MALDI仪器。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55254/55254fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55254/55254fig4.jpg" /> 图神经节苷脂对大鼠脑4. MALDI IMS。代表SourceÈMALDI IMS分子图像和DAN矩阵升华后在大鼠脑神经节苷脂的质谱。分子图像是重叠的频谱的多个神经节苷脂物种的复合物，并使用的Image J软件来显示整个大脑神经节苷脂物种的独特解剖学分布伪彩色。种GM1d18:1（红色，大众〜1547道尔顿），GM1d20:1（绿色，大众〜1573道尔顿），GM3d18:1（蓝，大规模〜1178道尔顿），以及GM3d20:1（水鸭，大众〜1207道尔顿）是表示。分析物的质谱显示离子丰度1000内 - 2000质量范围。主要的A系列神经节苷脂沿频谱标记。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55254/55254fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry at JoVE.com

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

DAN矩阵升华为神经节苷脂对大鼠脑组织的检测和可视化MALDI成像质谱

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

sublimation-dan-matrix-for-detection-visualization-gangliosides-rat

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

10.7K 次观看.  The University of Western Ontario. A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

视频: Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

注册生物成像，纳米材料可用于诊断和治疗监测

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

科研

生物工程

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

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI成像帕金森病的神经肽质谱

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

医学

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

鞘糖脂抗原的质谱分析

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

免疫与感染

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

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 ...

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 ...

生物学

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 ...

 Advanced MALDI-MSI Mapping of Metabolites in Brain Organoids

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry 

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge technology, allows us to map the spatial distribution of diverse molecules such as lipids, sugars, amino acids, drugs, and their metabolites within these organoids, all without the need for specific molecular probes. High-quality MSI data hinge on meticulous sample preparation. Fixatives play a pivotal role, but conventional options such as glutaraldehyde, paraformaldehyde, and cryopreserving such as sucrose may inadvertently impact tissue metabolites. Optimal fixation entails flash freezing in liquid nitrogen. However, for small organoids, a more suitable approach involves transitioning the organoids directly from the incubator into a warmed embedding solution, followed by freezing in dry ice-cooled ethanol. Another critical step is the embedding prior to cryosectioning, which also requires materials compatible with MSI, as traditional options can interfere with matrix deposition and ionization. Here, an optimized protocol for high resolution-MALDI-MSI of human brain organoids is presented, encompassing sample preparation, sectioning, and imaging using mass spectrometry. This method showcases the molecular distribution of small metabolites, such as amino acids, with high mass accuracy and sensitivity. As such, coupled with complementary studies of brain organoids, it can assist in illuminating complex processes governing early brain development, metabolic cell fate trajectories, and distinctive metabolite signatures. Furthermore, it provides insights into the precise locations of molecules within the organoid, enriching our understanding of the spatial organization of 3D brain organoid models. As the field continues to advance, a growing number of studies leveraging MSI to delve into brain organoids and complex biological systems is anticipated, thereby deepening the understanding of the metabolic aspects of human brain function and development.

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

An advanced method was developed for mass spectrometry imaging (MSI) of brain organoids that allows mapping metabolite distributions within these models. This technology offers insights into brain metabolic pathways and metabolite signatures during early development and in disease, promising a deeper understanding of the human brain function.

DAN矩阵升华为神经节苷脂对大鼠脑组织的检测和可视化MALDI成像质谱

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Molecular Imaging of Human Brain Organoids Using Mass Spectrometry