我们感谢汉娜华纳贡献数据的宝贵意见，为图3和乔纳斯贝格斯教授。瑞典研究理事会（格兰特522-2006-6416（马），521-2007-5407（马）;“阿克Wiberg基金会”（MA，歼轰），瑞典皇家科学院（硕士，JH）和瑞典化学感谢协会（JH）的财政支持。

该协议是调整多个大鼠脑部分，一般为20-30节的MALDI IMS数据统计分析的目的，由五个不同的步骤，包括组织准备，矩阵应用，MALDI-TOF质谱分析，数据评估和神经肽鉴定。概述和更详细的下面所述的程序： 1。组织编制此过程包括收集各自的组织样本，以及组织切片IMS的分析。蛋白质和多肽分析中的一个特定的目标，是为了避免降解。因此，在组织解剖的快速和勤奋是必不可少的工作。 <ol><li>牺牲斩首大鼠（一般为250-300克），粉状干冰&lt;30岁和冻结时间内最大的验尸报告中删除，然后转移到-80°C冰箱大鼠脑组织。使用液氮的更快冻结增加脑组织，这将产生负面影响矩阵结晶microtears的风险，从而减少MS的质量（图2D）。整个大脑可以存储几年前没有MS的信号质量损失切片。 </li><li>削减12微米切片和解冻贴装组织切片在导电的电离玻璃幻灯片（铟锡氧化物涂层幻灯片，布鲁克道尔顿）或MALDI靶（图2A-C的）在低温恒温器切片冰冻组织。 </li><li> 15分钟，在真空条件下，商店幻灯片干节，在​​-80°C，直到进一步的使用。应在最短的时间内分析组织切片，切片后，即使在-80°C。我们发现，在储存一年后，MS信号质量将明显减少。为了减少氧化蛋白质和多肽，储存容器中的空气可以更换的惰性气体（如氩气或氮气）。 </li></ol> 2。矩阵的应用矩阵的应用步骤有频谱质量产生重大影响，并要求组织以及分析物取决于多个参数的优化。这些因素包括化学参数，如一种基质，基质浓度，pH值，组织清洗和有机改性剂以及包括存款量，横向分辨率和证言10（图2D）设置。对于大规模的实验，这是减少例如方差，应用矩阵的所有部分，并在一天之内由同一运营商的重视。虽然有许多的策略申请如升华或矩阵小水滴阵列的自动沉积，喷雾，大小约100-150微微升，已成功地用于分析在各种组织中的小分子蛋白和神经肽的矩阵解决方案，包括大脑的第9，10,11，12，13。 <ol><li>解冻的部分为1小时在干燥器我们的。 </li><li>确保实验是由一个人以外的其他经营者所蒙蔽。重新标签的所有样品。 </li><li>洗节1X在70％乙醇（乙醇，在室温下，RT）为10秒和10秒两次在95％的乙醇（RT）的。对于大型实验，进行清洗所有玻璃幻灯片一起使用试管，以最大限度地减少变异。 </li><li>干燥器在10分钟的部分擦干。 </li><li>评估组织切片，在显微镜下检查组织失真，的microtears和细小裂纹，这将损害MALDI质谱质量（图2D）。 </li><li>准备好新鲜基质的解决方案，50％甲醇，10％150MM醋酸铵（AMAC）和0.3％三氟乙酸（TFA），水在50毫克/毫升道亨银行组成。 </li><li>矩阵的应用进行离散雾滴沉积在一个长方形的使用化学喷墨打印机的模式（芯片，岛津）。第一步是优化肽分析includi的矩阵应用的实验参数纳克每通液滴，传球次数。这个实验是由申请多个不同的应用参数矩阵阵列上相同的组织切片，同时确保每个阵列覆盖类似的大脑区域，如胼胝体，皮层和纹状体。进行相同的实验，每次参数变化，包括不同的大脑结构，针对特定分析物的不同矩阵，如果需要具体分析物提取不同基质溶剂。 </li><li>扫描组织切片与玻片架，对准持有人。组织切片上的定义为矩阵申请您的阵列和指定的空间分辨率，即当场发现的距离。适用于矩阵上使用的化学喷墨打印机的优化协议。对于这个实验，我们用以下的印刷参数的肽成像的优化协议：10滴（100 PL /下降），10应用程序传递和现场发现ð300微米istance的。 </li><li>最后的矩阵扫描发现部分和登记保存的图片前MALDI数据采集（步骤3.4）。 </li><li>进一步利用干燥器在真空状态下，直到存储的部分。 </li></ol> 3。 MALDI MS数据采集与处理 MS分析神经肽的电离时间飞行仪表（的UltraFlex二），布鲁克道尔顿，德国在反射模式经营，使用软件辅助数据采集，从每一个矩阵点14。因此准确的空间教学势在必行。重要的是电离优化，收购，特别是目标登记实验是由同一经营者，最好应蒙蔽实验组进行。电离的实验与多个载玻片的大规模实验，可以通过一个运营商，而另一人是经营化学喷墨打印机。 <ol><li>装入质谱仪玻片。 </li><li>检查电离采集方法使用一种低分子量的标准校准混合（布鲁克道尔顿）的校准。 </li><li>优化采集参数。 <ol><li>为了优化MS信号，避免烧蚀从周边矩阵存款矩阵，激光和组织的最佳焦点的大小应确定。 </li><li>激光能量设置，以确保尽可能多的矩阵存款最大的MS质量没有提升基准，减少峰值分辨率或饱和探测器。 </li><li>评估现场拍摄每矩阵的最大数量，直到唯一的噪音检测，往往一○○○年至2000年拍摄。估计应积累和激光位置之前获得在现场拍摄的数量应改变拍摄的数量。以每个矩阵点均匀采样，累积600杆25杆的步骤总数为24个步骤，使用RANDOM运动模式，从每个基质沉积。 </li></ol></li><li>注册的所有发现的部分电机坐标的电离阶段使用的FlexImaging（2.0版）10扫描和执行由AutoXexuteBatchRunner.exe软件在批处理模式下的数据采集。 </li><li>每个基线减法（凸包V3），平滑和外部校准（可选），其次作为一个ASCII文件（*。DAT，*。txt或*。csv格式）由出口加工单谱15。 </li></ol> 4。数据评估最终的数据评估，包括重点只在高峰期的信息，统计分析，数据后处理和数据减少。 <ol><li>作为第一步，电离IMS部分overnormalization效果进行了评价。这可以很容易地实现采用如FlexImaging（布鲁克·道尔顿）或BioMap（诺华）的数据可视化工具。作为第一步，总离子图像EVAluated前总离子流图（TIC）正常化，其次是单离子的各种突出肽峰的分布图像的人工检查。为特征峰的强度分布看，如果他们与组织功能（损害赔偿），发现质量或正常化的影响（图3）。 </li><li>根据组织学特征划定地区的利益（如纹状体）和ASCII文件格式导出相应的光谱。最好可以在这个阶段，谱的总离子流图（TIC）的正常化。 </li><li>导入ASCII文件，作为原产地等数据处理软件（v.8.1 Originlab），MATLAB（MathWorks公司，Natick市，马萨诸塞州，美国）或R 16。峰值检测可以使用高峰发现在软件附带的工具，例如在原产地或“mspeaks”在Matlab的“高峰分析”。作为一个单一的制表符分隔的文本文件，导出所有光谱的peaklists。 </li><li>以确定检测肽斌边界峰，进行分级分析，使用适当的软件工具（如pbin 17）或内部书面MATLAB或R脚本在这里单一的文本文件，其中包含所有的高峰采摘的数据加载到软件，为繁忙的边境决心指定参数等多久高峰应该以有关实验在光谱。例如，在实验中包含动物2组，每组5只动物，100光谱收集从每个动物和地区的利益。假设一个高峰可能是有趣的，如果它是目前在至少在一组（3/5），并在这些动物的光谱（3x50 = 150谱）中至少有一半的大多数动物，这将使总的百分比15％，总投资1000（2x5x100）光谱150正面的光谱。使用pbin工具，这一步产生一个单一的binrange文件，其中包含所有的bin从采集到的数据确定的宽度。为了验证斌边界是适当的，它是很简单的可视化与原始光谱的痕迹，在原产地的垃圾箱。 </li><li>峰面积积分可以降低方差统计分析是非常重要的。我们写脚本内​​部使用的R之间的高峰在第4步确定边界曲线下的面积来计算。综合峰地区进口到MS Excel（v.2007）和统计分析，非参数未成测试使用SAM工具执行18。 </li></ol> 5。多肽鉴定观测到的肽序列身份验证是必要的，以完成生物相关。最准确的方法包括直接关闭使用串联质谱（MS / MS），组织多肽碎片真正的自上而下的决心，虽然这种分析12,13肽浓度高。对于低丰度的肽或CLOS多肽EM / z值（±0.5％），组织分析减值和利用，包括提取，分离和基于MS的内源性神经肽鉴定小康的组织分析使用peptidomic战略。这里介绍的实验，中央重点是阿片肽的检测，这是一个特别的挑战，因为这些肽是相当低丰度相比，与其他神经肽​​谱。此外，这些肽是而极，这使得它们相对亲水性，很难留住共同肽的提取和分离技术......因此，我们采用基于标准的LC-MS/MS肽识别9,19组合组织提取和阿片肽的初步分离了以前报告的协议。 <ol><li>收集感兴趣的目标结构（伏隔核，NAC;尾壳核，CPU）的冠状部分。在低温恒温切片装入冷冻大鼠脑和消除周围的脑材料（CORTEX，隔，胼胝体用手术刀）。收集部分（30微米; N = 50）的解剖NAC和CPU和解冻安装NAC和CPU部分的章节不同的玻片上。 </li><li>加入100μL5％ACN/0.1％TFA的，两分钟孵育提取了组织的肽，并收集在离心低蛋白结合管。重复此步骤两次。 </li><li>执行由强阳离子交换色谱手段逐步在离子强度增加19（N = 4）洗脱肽的初步分离。 Speed​​Vac浓缩真空下使用干下来的样本。 </li><li>肽组分分析手段（1100年，安捷伦科技公司，加利福尼亚州圣克拉拉）纳流C18的反相液相色谱串联质谱法（LC-MS/MS）接口。 MS实验进行混合的线性iontrap /傅里叶变换离子回旋共振（FTICR）仪，Thermo Scientific的LTQ FT 7T，沃尔瑟姆，马，肽fulscan光谱（m / z为150-2000）被收购FTICR随后的5肽在iontrap最激烈的碰撞诱导解离（CID）9峰碎片分析与质量分辨率高（100000）。 </li><li>肽序列鉴定数据库匹配，并可以通过补充的从头测序分析。数据库检索，市售的搜索引擎（吉祥物，XTandem或蛋白质探矿）雇用20。通常对含有已知或预测的神经肽和神经肽前体蛋白21的序列序列数据库进行搜索。 </li></ol> 6。代表结果根据协议，导致1000多峰检测约300个分子种类单一同位素（平均光谱显示相应编制MALDI成像纹状体组织切片质谱图。 1）。使用Flex成像软件实现数据可视化突出的分子离子峰和显示特征峰的强度分布，解剖特征（图3）。一个进一步的MALDI IMS的特点是其相对的重复性好。在这个实验中，所有检测到的分子种类的峰值强度的方差系数为30％，但许多山峰显示非常低的变化和治疗组内的高重复性（图4）。四个不同地区的利益，包括背外侧和背内侧部分损毁和完整芨相对峰强度数据进行统计分析。为了调整同时多重比较，统计分析，非参数测试手段使用SAM工具18。最突出的变化，发现在失神经的多巴胺，帕金森病纹状体背外侧部分。这里SI<html>在gnificant不同的治疗组之间的变化，观察两个强啡肽，强啡肽B和α-neoendorphin的（图5）。详细，两强啡肽峰强度相对增加50-60％，高的运动障碍动物观察比较低的运动障碍动物和损伤控制（P &lt;0.05 F（2，15）= 12.8 DynB，= 5.7 aNeo的;图5）。 <img alt="图1" src="/files/ftp_upload/3445/3445fig1.jpg" /> 图1平均的MS跟踪获得两个密切相关的地区纹状体尾壳核（CPU）和伏隔核（NAC）。两个区域显示在一个地区独特的表达了一些不同的分子种类的MS型材，或在不同的峰值强度级别（插入，m / z为2028）。可以使用专门的图像处理软件（底部面板）可视化的空间分布格局，各峰。 2.JPG“/&gt; 图2（一）大脑在低温恒温器使用嵌入媒体（场外交易市场;箭头）夹头安装。，护理是采取场外不污染的大脑区域，是因为场外原因离子抑制肽 ​​切片。 （乙，丙）薄片（约12微米厚）安装到电离兼容玻片解冻，干涸了几秒钟，以避免冻害，在C（ð）Microtears的可能是肉眼难以察觉，但损害的MALDI基质的结晶，泯的MALDI质谱信号。相同的部分用甲酚紫染色揭示microtears和裂缝（右下显微照片）。 <img alt="图3" src="/files/ftp_upload/3445/3445fig3.jpg" /> 图3中的数据进行评估的第一步是进行可视化分析（人工智能）的质量范围跨越几个不同的峰。在这里，从9只小鼠的纹状体部分与MALDI质谱成像。可视化的平均总我电流将揭示突出的高或低离子强度（箭头）的地方。受到这些地区可以超过或低于正常化的影响和扭曲的数据分析，妥协的结果。差峰值分布解剖定义，揭示普遍较低的峰值信号与噪声的部分，例如第3和第9条，通过一峰F <img alt="图4" src="/files/ftp_upload/3445/3445fig4.jpg" /> 图4。MS治疗组之间的重复性，可以通过计算平均MS跟踪，并为每个m / z值（插入，m / z为722和1749）的标准错误评估。重现性好，确保有效的统计分析。 <a href="http://www.jove.com/files/ftp_upload/3445/3445fig5_lg.jpg"><img alt="图5" src="/files/ftp_upload/3445/3445fig5.jpg" /></a> 图5。强啡肽B和α-neoendorphin的峰强度显着增加，在6-OHDA-损毁，帕金森病，运动障碍动物纹状体（高清;箭头）相比，以低运动障碍（LD）和病变的对照组（LC）。肽峰值强度平均褶皱变化保持不变的一面±SEM（病灶/健侧）表示。 * P &lt;0.05; CX皮层; CC，胼胝体。比例尺5毫米。

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L., Chaurand, P., 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=MALDI+imaging+mass+spectrometry:+molecular+snapshots+of+biochemical+systems.">MALDI imaging mass spectrometry: molecular snapshots of biochemical systems.</a> Nat. Methods. 4, 828-833 (2007).</li><li>Ljungdahl, <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+Reveals+Elevated+Nigral+Levels+of+Dynorphin+Neuropeptides+in+L-DOPA-Induced+Dyskinesia+in+Rat+Model+of+Parkinson's+Disease.">Imaging Mass Spectrometry Reveals Elevated Nigral Levels of Dynorphin Neuropeptides in L-DOPA-Induced Dyskinesia in Rat Model of Parkinson's Disease.</a> PLoS ONE. 6, e25653 (2011).</li><li>Groseclose, M. R., Andersson, M., Hardesty, W. M., 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=Identification+of+proteins+directly+from+tissue:+in+situ+tryptic+digestions+coupled+with+imaging+mass+spectrometry.">Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometry.</a> J. Mass. Spectrom. 42, 254-262 (2007).</li><li>Andersson, M., Groseclose, M. R., Deutch, A. Y., 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=Imaging+mass+spectrometry+of+proteins+and+peptides:+3D+volume+reconstruction.">Imaging mass spectrometry of proteins and peptides: 3D volume reconstruction.</a> Nat. Methods. 5, 101-108 (2008).</li><li>Deininger, S. -. O., Setou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging Mass Spectrometry. , 199-208 (2010).</li><li>Norris, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+MALDI+Mass+Spectra+to+Improve+Mass+Spectral+Direct+Tissue+Analysis.">Processing MALDI Mass Spectra to Improve Mass Spectral Direct Tissue Analysis.</a> Int. J. Mass. Spectrom. 260, 212-221 (2007).</li><li>Ihaka, R., Gentleman, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Language+for+Data+Analysis+and+Graphics.">A Language for Data Analysis and Graphics.</a> Journal of Computational and Graphical Statistics. 5, 299-314 (1996).</li><li>Tusher, V. G., Tibshirani, R., Chu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+analysis+of+microarrays+applied+to+the+ionizing+radiation+response.">Significance analysis of microarrays applied to the ionizing radiation response.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 5116-5121 (2001).</li><li>Bergstrom, L., Christensson, I., Folkesson, R., Stenstrom, B., Terenius, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ion+exchange+chromatography+and+radioimmunoassay+procedure+for+measuring+opioid+peptides+and+substance+P.">An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P.</a> Life. Sci. 33, 1613-1619 (1983).</li><li>Falth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptidomics+strategies+for+specific+and+sensitive+identification+of+endogenous+peptides.">Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides.</a> Mol. Cell. Proteomics. 6, 1188-1197 (2007).</li><li>Falth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SwePep,+a+database+designed+for+endogenous+peptides+and+mass+spectrometry.">SwePep, a database designed for endogenous peptides and mass spectrometry.</a> Mol. Cell. Proteomics. 5, 998-1005 (2006).</li></ol>

作者没有透露。

有用人中的神经肽的研究MALDI成像质谱仪的几个优势。一个公正的MS数据分析，可以揭示只有特定的脑细胞，或在这里只有纹状体背外侧部分具有一定的病理生理条件的结果。所保留的空间信息，然后就可以重新定义地区的利益，执行更高的灵敏度和较低的变异相比，全脑切片或使用传统peptidomics肽提取物的研究与分析统计分析。此外，重要的是实现电离的IMS随时可以发现以前未知的翻译后修饰，但必须遵循结构分析，以确定确切的氨基酸被修改的位置。 常见的陷阱，在可视化的MALDI IMS数据包括映射的最大峰值强度BL线性光学尺ACK（0％），每节实验系列（图3），而不是所有的部分映射到一个共同的绝对规模，其中100％，是所有路段的最大峰值强度，色彩（100％）（图5） 。后一种方法允许组数据和可视化治疗组之间的差异比较。 电离IMS的分析中的一个主要障碍是分配到特定的质量峰的肽。组织串联质谱有时是可能的，但常常被证明是相当困难的13,14。我们发现一个更传统的方法，包括对强阳离子交换层析，反相LC-MS/MS分析，制备分馏可以用来成功许多神经肽序列，尤其是阿片肽。获得质量好MS / MS谱图，不匹配任何数据库中的条目，使用常用的搜索引擎，如福星，它仍然是并不鲜见。在这些情况下，手从头测序是ONLy选项。最终证明高峰身份可以通过MALDI IMS从相应的基因剔除小鼠的组织切片，但这并不总是可用的或可能的。另一种方法是由西方免疫或免疫组化的例子，来验证结果截然不同的方法。这通常可以包括提高抗体和相当数量的工作，验证新的抗体。 在本议定书提出的总体战略，为大型神经肽的MALDI IMS实验，其中包括几个部分和实验条件进行了优化。该协议已得到优化，尤其是阿片肽，并在未来的研究将有很大的影响，在不同的研究领域包括机制的疼痛和内源性药物成瘾就业。

1.1样品采集和准备<ol><li>异氟醚（Apoteksbolaget AB，瑞典） </li><li>手术工具;两对剪刀，咬骨钳，压舌板</li><li>精细粉碎的干冰</li><li>铝箔塑料袋和存储</li><li>杜瓦（MICROM，德国海德堡） </li><li>铟锡氧化物（ITO）涂层电离幻灯片（布鲁克道尔顿，德国） </li><li>最佳切削温度的介质（OCT）（樱花Finetek欧洲，荷兰） </li><li>真空干燥器</li></ol> 1.2矩阵的应用<ol><li>乙腈（ACN），甲醇（甲醇），乙醇（酒精）亲分析级（默克公司，德国）。 </li><li>三氟乙酸（TFA）（Sigma Aldrich公司，圣路易斯，密苏里州）。 </li><li>纯化水用的Milli-Q净化系统（Millipore公司，贝德福德，马萨诸塞州）。 </li><li>芯片1000（日本岛津）或纵向630去污剂（LabCyte，桑尼维尔，CA） </li></ol> 1.3质谱<ol><li> UL二traflex（布鲁克道尔顿） </li><li> FlexImaging软件（V 2.0，布鲁克道尔顿） </li><li>肽或蛋白质的标定标准（布鲁克道尔顿） </li></ol> 1.4数据处理<ol><li>原产v.8.1（OriginLab公司，Norhampton，马） </li><li> Matlab版本7.9.0.529（MathWorks公司，Natic​​k市，马） </li><li> pBin（ <a href="http://www.vicc.org/biostatistics/software.php" target="_blank">http://www.vicc.org/biostatistics/software.php</a> ） </li></ol> 1.5神经肽鉴定<ol><li>超声sonicator </li><li> 10 kDa的分子量截止滤波器（Millipore公司，贝德福德，马萨诸塞） </li><li> SP葡聚糖Ç25凝胶（Sigma公司，德国） </li><li> HYPERSIL列（ThermoFisher） </li><li>吡啶，甲酸（FA）乙腈（ACN），甲醇（甲醇），乙醇（酒精）的亲分析级（默克公司，德国）。 </li><li>三氟乙酸（TFA），Sigma Aldrich公司（圣路易斯，密苏里州，美国）。 </li><li>安捷伦1100纳流LC，microWPS，C18柱（0.075x150mm） </li><li>安捷伦1100微fractioncollector的，Prespotted anchorchip目标板（布鲁克道尔顿） </li><li> BioTools软件（至五3.1 SR2中，布鲁克·道尔顿） </li><li>生物工程软件（v.3.3，赛默飞世尔; <a href="http://www.thermoscientific.com" target="_blank">www.thermoscientific.com</a> ） </li><li> Mascot软件（V 2.2，MatrixScience; <a href="http://www.matrixscience.com" target="_blank">www.matrixscience.com</a> ） </li><li> swepep数据库（ <a href="http://www.swepep.org" target="_blank">www.swepep.org</a> ） </li></ol>

maldi imaging mass spectrometry of neuropeptides in parkinson's disease

MALDI成像质谱（IMS）是一个功能强大的方法，有利于分子物种在生物组织样品2（图1）的空间分析。 12微米的薄组织切片覆盖，这有利于解吸和完整的多肽和蛋白质，可以检测与质量分析仪，通常使用MALDI TOF / TOF质谱仪的电离与电离矩阵。一般几百峰，可以在一个单一的大鼠脑组织切片评估。中常用的成像技术相比，这种方法并不需要先验知识感兴趣的分子，并允许无监督和综合分析多种分子物种，同时保持高的分子特异性和灵敏度2。在这里，我们描述了1电离基于IMS的方法，阐明在动物模型的帕金森氏病（PD）大鼠脑特定区域的神经肽的分布状况。 PD是一种常见的神经退行性疾病与3,4岁以下65人以上的1％的患病率。最常见的对症治疗的基础上多巴胺替代使用左旋多巴5。然而，这是伴随着严重的副作用，包括不自主的异常运动，称为左旋多巴诱发的运动障碍（盖）1,3,6。盖分子的最突出的变化之一是阿片类易制毒化学前强啡肽mRNA的上调。强啡肽调节神经递质在大脑基本上是在运动控制7,8涉及的领域。然而，迄今为止尚未从神经肽前体的加工确切的起源阿片肽特点。因此，我们利用电离的IMS实验帕金森氏症和左旋多巴引起的运动障碍的动物模型。 MALDI成像质谱被证明是特别有利的方面肽characterizaTION，因为常用的抗体为基础的方法，针对已知的多肽序列和先前发现的翻译后修饰。 IMS的电离通过对比可以揭示新型肽加工产品，从而揭示神经肽的神经传输调制的新的分子机制。肽离子的相对丰度，而神经肽的绝对数额不能确定用MALDI IMS，可以划定质谱，提供有关健康和疾病的变化水平的见解。在这里介绍的例子中，被发现的，α-neoendorphin的强啡肽B和P物质的峰强度显着增加在背侧，但不背内侧纹状体的动物有严重运动障碍累及面部，躯干和orolingual肌肉（图5）。此外，IMS的电离透露，运动障碍的严重程度和DES-酪氨酸α-neoendorphin的水平之间的关系，代表的功能inacti的以前未知的机制VATION减少在去除N-端酪氨酸纹状体强啡肽强啡肽的阿片受体结合能力9。这是第一个研究神经表征中使用MALDI IMS和业绩突出的技术在生物医学研究的各个领域的应用潜力的盖子。

Watch this Scientific Journal Video about MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease at JoVE.com

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

使用左旋多巴的多巴胺替代药物治疗是最常用的对症治疗帕金森氏病，但伴随着不自主的异常运动，被称为运动障碍的副作用，包括<sup&gt; 1</sup&gt;。在这里，协议为MALDI成像质谱检测大鼠脑组织中神经肽水平相关的运动障碍的变化。

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 ...

maldi-imaging-mass-spectrometry-neuropeptides-parkinson-s

Research

JoVE Journal

Medicine

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

25.8K 次观看.  Uppsala University. 使用左旋多巴的多巴胺替代药物治疗是最常用的对症治疗帕金森氏病，但伴随着不自主的异常运动，被称为运动障碍的副作用，包括 1。在这里，协议为MALDI成像质谱检测大鼠脑组织中神经肽水平相关的运动障碍的变化。

视频: MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

1单侧损毁6  - 羟基多巴胺帕金森病小鼠模型的发展

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

科研

医学

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

推导人类纤维原细胞的皮肤穿刺活检植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

基因-环境相互作用模型揭开帕金森病的隐形易感机制

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

控制帕金森氏症与自适应深部脑刺激

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

在脑组织成像金属激光剥蚀 - 电感耦合等离子体 - 质谱法（LA-ICP-MS）

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

解剖性脑 MRI 的全脑分割及变化点分析--Premanifest 亨廷顿病的应用

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...