Name: optimal preparation of formalin fixed samples for peptide based matrix assisted laser desorption/ionization mass spectrometry imaging workflows
Uploaded: 2018-01-16T14:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows at JoVE.com

作者要感谢悉尼医学院基金会和布鲁斯和基金会的资助, 这项工作的一部分, 通过他们的博士奖学金计划的阿尔茨海默病研究和 ARC 发现补助金 (DP160102063) 授予 PKW。

警告: 执行此过程时应遵循所有适用的安全措施, 包括使用适当的个人防护设备 (PPE) (例如,实验室大衣、腈手套、安全眼镜、等)
1. 试剂和设备的制备
<ol><li>
解决方案的准备
	<ol>
<li>准备500毫升的 Carnoy 的液体, 混合100% 乙醇 (乙醇), 氯仿和冰醋酸在一个 6:3: 1 伏/v/v 的比例在一个干净的玻璃肖特瓶。</li>
		<li>使液体硝化纤维 (nc), 溶解在整齐的丙酮的 nc 膜 (通常用于西部印迹), 由温和的搅拌, 最终浓度为40毫克/毫升。</li>
	</ol>
</li>
	<li>
数控涂布滑轨的研制
	<ol>
<li>使用类似于用于组织学检查的血液涂片制备的技术20准备 NC 涂层幻灯片。</li>
		<li>在导电铟锡氧化物 (ITO) 显微镜滑动的一条边上, 将液体 NC 40 µL。使用常规的玻璃显微镜滑动, 在滑动的表面张力下拖动 NC 以创建均匀的薄膜涂层。</li>
		<li>允许 NC 在室温下干燥二十年代, 然后在室温下存储直到需要。在这些条件下的样品储存可以是无限期的, 只要组织保持和没有灰尘。 注: 这种制备方法更被称为 "薄膜", 并将产生一种均匀的涂层, 它依赖于 NC 的粘度以及其表面张力在 self-level 上的缺乏。在准备幻灯片时, 唯一需要注意的是不要移动得太快, 这将创建 NC 的条纹而不是完整的覆盖范围。 注意: 由于液态 NC 干燥得非常快, 所以必须迅速工作以避免溶液在适当扩散之前干燥。在处理 NC 时也应注意其液态状态, 因为它是一种高能的化合物, 不应与点火源接触以防止燃烧爆炸。NC 在干燥过程中也会发生吸热转变, 如果允许长时间接触皮肤或戴手套的手, 则会导致冷灼伤。为了尽量减少所有相关风险, 每次只应准备少量 (< 1 毫升建议)。一旦干燥, 它在室温下是稳定的。但是, NC 在大量存在时仍能保持爆炸性。</li>
	</ol>
</li>
	<li>
定制蒸气室的创建
	<ol>
<li>切一张厚的纸, 一把剪刀大到足以适合底部一半的标准塑料培养皿 (直径94毫米和3毫米厚)。</li>
		<li>切纸, 在中心留下一个长方形的长条, 与显微镜幻灯片一样大小, 留下足够的过剩, 使纸张将保持其在培养皿中的位置 (补充图 1)。</li>
	</ol>
</li>
</ol>2. 组织切片的制备
<ol><li>
FFPE 组织21。
	<ol>
<li>选择所需的组织块和它在12µm 厚度在标准的长凳上安装切片和浮动安装到 NC 预 ITO 幻灯片。</li>
		<li>将新鲜的二甲苯中的玻片浸入, 去除剩余的石蜡2分钟, 再重复一次操作。 注: 如果石蜡块特别普遍或组织相对于石蜡块比较小, 样品可以在60° c 加热, 以在二甲苯洗涤前融化大多数。</li>
		<li>洗涤 deparaffinized 样品通过淹没幻灯片在一个分级的溶剂系列: 70% 伏/v 乙醇/水, 100% 乙醇, Carnoy 的液体, 100% 乙醇, 超纯水和100% 乙醇。每个浸没的持续时间应该是三十年代, 除了 Carnoy 的流体, 必须持续2分钟。 注意: Carnoy 的液体和二甲苯必须储存在通风柜中使用, 因为它们在吸入时具有剧毒。</li>
	</ol>
</li>
	<li>
福尔马林固定冷冻 (FFF) 组织 注: 样品应已冻结, 并根据样品类型适当。<ol>
<li>平衡在切片的工作温度下的组织块进行20分钟的22。 注意: 平衡温度取决于组织的类型。</li>
		<li>节的组织使用切片在12µm 和解冻安装的部分到准备 NC 涂层 ITO 幻灯片。</li>
		<li>将幻灯片放在黑暗中的长凳上的真空干燥里。 注: 在这些情况下, 样品可以存放数周。</li>
		<li>按 FFPE 组织 (2.1.3) 所述的分级溶剂系列清洗样品。 注: 不需要二甲苯 deparaffinisation 步骤, 因为样品没有嵌入。</li>
	</ol>
</li>
</ol>3. 亚甲基交联水解
<ol><li>将示例幻灯片 (FFF 或 FFPE) 装入一个塑料滑动框, 其中填充有20摩尔三盐酸 (pH 8.8)。密封的盒子, 并把它放在一个水浴, 包含500毫升的水, 让盒子触摸底部。然后加热15分钟, 在120° c 的压力锅, 可以达到70人民军的操作压力。</li>
	<li>取出幻灯片, 让它们冷却, 让它们在室温下干燥15分钟。</li>
</ol>4. 组织消化
<ol><li>一旦水解, 用10µL 的胰蛋白酶溶液 (1 毫克/毫升) 在超纯水中涂上样品, 通过移10µL 溶液到组织切片的边缘, 然后使用同样的吸管尖端, 将液滴穿过表面的组织的整个表面。紧张. 注意: 避免用吸管尖端刮伤组织表面, 并在组织边缘外扩散酶。</li>
	<li>允许样品在室温下干燥。</li>
	<li>一旦干燥, 将样品装入先前构造的蒸气室的顶部 (组织朝下), 在幻灯片的任一边缘 (补充图 2) 使用高压釜胶带。</li>
	<li>吸管600µL 的解决方案, 其中包含100% 乙腈和50毫米碳酸氢铵的混合物仔细到印迹纸手指在培养皿的底部, 直到手指似乎是均匀湿润。</li>
	<li>将蒸气室的上半部分放在下半部, 确保纸手指和样品滑动对齐, 并将其密封在其赤道周围与石蜡膜。</li>
	<li>在37° c 的恒温箱中过夜, 让样品完全消化。</li>
</ol>5. 通过升华的基体涂层
<ol><li>一旦消化, 在5位数的分析天平上称量样本幻灯片。</li>
	<li>将滑块安装在升华装置的冷却手指上, 用铜带固定, 与蒸汽室 (补充图 3) 所描述的方法相同。 注意: 为确保滑块与冷却手指均匀接触, 将一层铜带放在冷却手指的底部, 使其表面平整。这保证了滑动均匀地被冷却, 导致甚而基体的证言。</li>
	<li>将α-氰基 4-肉桂酸 (CHCA) 基体的300毫克放入腔室底部的玻璃培养皿中, 均匀分布, 形成一层薄薄的基体晶体。</li>
	<li>组装的升华和安全的两个半马蹄形钳。将组装单元悬挂在一个沙浴上方15-20 厘米处, 预热于220° c, 将其放置在与反击架相连的金属环上。</li>
	<li>将腔室连接到真空源, 接触真空, 使其稳定下来至〜 25 mTorr 5 分钟。</li>
	<li>用冰把冷却手指放到顶部, 加50毫升的水。允许仪器在继续前5分钟再解决。</li>
	<li>将腔室放在沙子的表面上, 确保沙子完全接触到腔室的底部, 并使其保持45分钟以创建理想的0.22 毫克/cm2的涂层。 警告: 在高真空处理玻璃器皿时应注意, 因为在疏散时, 设备受到的任何损坏都可能导致玻璃腔的灾难性故障。</li>
	<li>45分钟后, 通过提高金属环和排气室, 从砂浴中取出房间。 注: 一旦腔室被排出, 当空气中的水开始凝结在冻结的手指和样品滑动时, 就必须迅速地移除幻灯片。</li>
	<li>卸下滑轨并称重, 以确保所需的涂层已实现。 注: 如果涂层已达到不足, 只需重复升华过程的幻灯片, 直到所需的涂层厚度已经达到。</li>
</ol>6. 再结晶
<ol><li>一旦升华, 在先前建造的蒸气室的顶部安装试样滑动, 如前所述 (组织朝下)。</li>
	<li>吸管600µL 的溶液中含有 1:1 (v/v) 100% 乙腈和 0.1% v/v 乙酸酸在水中小心地涂抹在培养皿底部的印迹纸上, 以确保均匀的涂层。</li>
	<li>组装室, 确保纸张标签和显微镜滑动对准完美, 并留在37° c 孵化器1小时。 警告: 不要密封会议厅。 注: 一旦再结晶, 通常的黄色色调的矩阵应改为白色, 看起来不那么闪亮, 并出现非常均匀。这表明, 再结晶已正确执行。</li>
	<li>样品现在可以进行分析了。</li>
</ol>7. 仪器仪表
<ol><li>扫描平板扫描仪中的幻灯片以创建数字图像。</li>
	<li>将样品加载到质谱仪中, 然后使用相应的软件平台进行分析。</li>
	<li>分析了正离子 reflectron 模式的样品, 其质量范围为 750-3, 500 Da, 并对所需的结果进行了现场分辨。在大多数情况下, 20-50 µm 光栅宽度是充足的, 然而, 只需10µm 可以使用15。 警告: MALDI UV 激光器是电离辐射的来源, 不应直接查看。在操作前, 确保激光器包含在仪器屏蔽内。</li>
</ol>

<ol>
	<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>Caprioli, R. M., Farmer, T. B., Gile, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Biological+Samples:+Localization+of+Peptides+and+Proteins+Using+MALDI-TOF+MS.">Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI-TOF MS.</a> Anal Chem. 69 (23), 4751-4760 (1997).</li><li>Jackson, S. N., 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=MALDI-Ion+Mobility+Mass+Spectrometry+of+Lipids+in+Negative+Ion+Mode.">MALDI-Ion Mobility Mass Spectrometry of Lipids in Negative Ion Mode.</a> Analytical methods : advancing methods and applications. 6 (14), 5001-5007 (2014).</li><li>O'Rourke, M. 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+non-instrument-based+method+for+the+analysis+of+formalin-fixed+paraffin-embedded+human+spinal+cord+via+matrix-assisted+laser+desorption/ionisation+imaging+mass+spectrometry.">A non-instrument-based method for the analysis of formalin-fixed paraffin-embedded human spinal cord via matrix-assisted laser desorption/ionisation imaging mass spectrometry.</a> Rapid Commun Mass Spectrom. 29 (19), 1836-1840 (2015).</li><li>O'Rourke, M. B., Raymond, B. B. A., 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 (7), 637-644 (2015).</li><li>Chughtai, K., Heeren, 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=Mass+spectrometric+imaging+for+biomedical+tissue+analysis.">Mass spectrometric imaging for biomedical tissue analysis.</a> Chem Rev. 110 (5), 3237-3277 (2010).</li><li>Ye, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+mass+spectrometry-assisted+molecular+imaging+of+metabolites+during+nitrogen+fixation+in+the+Medicago+truncatula-Sinorhizobium+meliloti+symbiosis.">MALDI mass spectrometry-assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula-Sinorhizobium meliloti symbiosis.</a> Plant J. 75 (1), 130-145 (2013).</li><li>Powers, T. W., 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=MALDI+imaging+mass+spectrometry+profiling+of+N-glycans+in+formalin-fixed+paraffin+embedded+clinical+tissue+blocks+and+tissue+microarrays.">MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.</a> PLoS One. 9 (9), 10655 (2014).</li><li>Rompp, A., Spengler, B. <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+imaging+with+high+resolution+in+mass+and+space.">Mass spectrometry imaging with high resolution in mass and space.</a> Histochem Cell Biol. 139 (6), 759-783 (2013).</li><li>Shariatgorji, M., Svenningsson, P., Andren, P. E. <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+imaging,+an+emerging+technology+in+neuropsychopharmacology.">Mass spectrometry imaging, an emerging technology in neuropsychopharmacology.</a> Neuropsychopharmacology. 39 (1), 34-49 (2014).</li><li>Alexandrov, T. <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:+statistical+data+analysis+and+current+computational+challenges.">MALDI imaging mass spectrometry: statistical data analysis and current computational challenges.</a> BMC Bioinformatics. 13, 11 (2012).</li><li>Weaver, E. M., Hummon, A. B. <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:+From+tissue+sections+to+cell+cultures.">Imaging mass spectrometry: From tissue sections to cell cultures.</a> Adv Drug Deliv Rev. 65 (8), 1039-1055 (2013).</li><li>Watrous, J. D., Dorrestein, P. C. <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+in+microbiology.">Imaging mass spectrometry in microbiology.</a> Nat Rev Microbiol. 9 (9), 683-694 (2011).</li><li>O'Rourke, M., Padula, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Non-Instrument+Based+Preparation+of+Tissue+Samples+Destined+for+Imaging+Mass+Spectrometry+(IMS)+Analysis.">The Non-Instrument Based Preparation of Tissue Samples Destined for Imaging Mass Spectrometry (IMS) Analysis.</a> Protocol Exchange. , (2017).</li><li>O'Rourke, M. B., 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+new+standard+of+visual+data+representation+for+imaging+mass+spectrometry.">A new standard of visual data representation for imaging mass spectrometry.</a> Proteomics Clin Appl. 11 (3-4), (2017).</li><li>O'Rourke, M. B., 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=Analysis+of+formalin-fixed,+paraffin-embedded+(FFPE)+tissue+via+proteomic+techniques+and+misconceptions+of+antigen+retrieval.">Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval.</a> Biotechniques. 60 (5), 229-238 (2016).</li><li>Casadonte, R., 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=Proteomic+analysis+of+formalin-fixed+paraffin-embedded+tissue+by+MALDI+imaging+mass+spectrometry.">Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry.</a> Nat. Protocols. 6 (11), 1695-1709 (2011).</li><li>Ong, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry+Imaging+and+Identification+of+Peptides+Associated+with+Cephalic+Ganglia+Regeneration+in+Schmidtea+mediterranea.">Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea.</a> J Biol Chem. 291 (15), 8109-8120 (2016).</li><li>Seeley, E. H., 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=Molecular+imaging+of+proteins+in+tissues+by+mass+spectrometry.">Molecular imaging of proteins in tissues by mass spectrometry.</a> Proc Natl Acad Sci U S A. 105 (47), 18126-18131 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-smear+showing+Experimental+Infection+with+Herpetomonas.">Blood-smear showing Experimental Infection with Herpetomonas.</a> Proc R Soc Med. 18, 55 (1925).</li><li>Gorrie, C. 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=Effects+of+human+OEC-derived+cell+transplants+in+rodent+spinal+cord+contusion+injury.">Effects of human OEC-derived cell transplants in rodent spinal cord contusion injury.</a> Brain Res. 1337, 8-20 (2010).</li><li>Wu, Q., Comi, T. J., Li, B., 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=On-Tissue+Derivatization+via+Electrospray+Deposition+for+Matrix-Assisted+Laser+Desorption/Ionization+Mass+Spectrometry+Imaging+of+Endogenous+Fatty+Acids+in+Rat+Brain+Tissues.">On-Tissue Derivatization via Electrospray Deposition for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Endogenous Fatty Acids in Rat Brain Tissues.</a> Anal Chem. 88 (11), 5988-5995 (2016).</li><li>Sturm, R. M., Greer, T., Chen, R., Hensen, B., 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=Comparison+of+NIMS+and+MALDI+platforms+for+neuropeptide+and+lipid+mass+spectrometric+imaging+in+C.+borealis+brain+tissue.">Comparison of NIMS and MALDI platforms for neuropeptide and lipid mass spectrometric imaging in C. borealis brain tissue.</a> Anal Methods. 5 (6), 1623-1628 (2013).</li><li>Kompauer, M., Heiles, S., Spengler, B. <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+MALDI+mass+spectrometry+imaging+of+tissues+and+cells+at+1.4-mum+lateral+resolution.">Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-mum lateral resolution.</a> Nat Methods. 14 (1), 90-96 (2017).</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+sublimation/recrystallization+for+imaging+proteins+by+mass+spectrometry+at+high+spatial+resolution.">Matrix sublimation/recrystallization for imaging proteins by mass spectrometry at high spatial resolution.</a> Anal Chem. 83 (14), 5728-5734 (2011).</li><li>Rohner, T. C., Staab, D., Stoeckli, 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+mass+spectrometric+imaging+of+biological+tissue+sections.">MALDI mass spectrometric imaging of biological tissue sections.</a> Mech Ageing Dev. 126 (1), 177-185 (2005).</li><li>Li, B., Bhandari, D. R., Rompp, A., Spengler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+MALDI+mass+spectrometry+imaging+of+gallotannins+and+monoterpene+glucosides+in+the+root+of+Paeonia+lactiflora.">High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora.</a> Sci Rep. 6, 36074 (2016).</li><li>Spengler, B. <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+imaging+of+biomolecular+information.">Mass spectrometry imaging of biomolecular information.</a> Anal Chem. 87 (1), 64-82 (2015).</li><li>Widlak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+molecular+signatures+of+oral+squamous+cell+carcinoma+and+normal+epithelium+-+application+of+a+novel+methodology+for+unsupervised+segmentation+of+imaging+mass+spectrometry+data.">Detection of molecular signatures of oral squamous cell carcinoma and normal epithelium - application of a novel methodology for unsupervised segmentation of imaging mass spectrometry data.</a> Proteomics. 16 (11-12), 1613-1621 (2016).</li></ol>

作者没有利益冲突或商业利益透露

该协议旨在最大限度地电离分子物种的生成, 同时消除域的分析。在应用矩阵、消化样本或升华24后, 使用相同的覆盖原则是关键因素; 结晶。也就是说, 需要建立和维护一个均匀的蒸气、基体或其他的沉积物。移溶剂洗涤为再结晶和消化, 均匀地在样品之下, 防止任何单独区域接受更多溶剂蒸气比其他任何地方。这是重要的防止形成可见的冷凝和整体抑制域的表面分子, 以及确保样...

基质辅助激光解吸/电离质谱成像 (MALDI MSI) 已被用作图像技术的二十年1,2, 分析的生物分子范围包括: 脂类3, 多肽2 ,4,蛋白质2,5, 代谢物6,7, n-糖8, 以及合成分子, 如治疗药物 9,10。显示此技术实用工具的出版物数量在过去十年中显著增长6、11、12、13。某些分子, 如血脂, 是相对容易通过 MALDI MSI 分析, 因为它们电离容易由于其化学性质, 因此需要很少的前期准备3。然而, 对于更难的目标, 如肽, 有效电离这些分子所需的步骤是广泛的, 一般复杂的14。目前很少有出版物旨在处理或证明在使用这种独特的视觉技术的组织的方法15的重现性。出于这个原因, 我们已经将观察和实施的优化编译成一个单一的, 易于实施的方法, 不应该需要修改, 从甲醛交联组织来源的多肽的分析14。
在这份手稿中, 我们描述了一个验证的, 低成本的可重现性的方法, 用于检测和空间映射的多肽, 由福尔马林固定冷冻 (FFF) 和福尔马林固定石蜡嵌入 (FFPE) 组织切片。此方法不需要或依赖于任何专用的工具3。具体来说, 我们处理的许多方面的专业样品准备必要的分析多肽;步骤, 如抗原检索16和基体涂层。我们的协议还利用了廉价的设备和试剂, 从而使这种方法可供更广泛的社区使用, 否则将无法负担替代的机器人设备17。
开发人工样品制备方法的背后的原因是两个方面: 第一, 使用升华创建一个一致的和均匀的矩阵晶体的涂层, 是〜1µm 长度为18, 这是无法做到的, 更常见的喷涂技术.其次, 相对较小的设置成本: 自定义设备的总成本为 < 1500 澳元。我们注意到, 在成本效益方面, 当没有机器机器参与的时候, 每个样品的价格就会便宜得多。以前已经报告过升华的使用, 然而, 据我们所知, 在文献中没有报告也没有描述这一过程和样品制备的 step-by 步骤方法。
本协议旨在帮助研究人员获得 MALDI 质谱仪, 并意图生成与感兴趣的生物分子相关的空间信息19。在本质上, MALDI MSI 是一种组织学检查的形式, 不依赖于抗体或污点2。

<table><tbody><tr><td>Cryo Microtome</td><td>Leica</td><td>CM3050</td><td>For preparation and section of tissue.</td></tr><tr><td>Indium Tin Oxide Microscope slides</td><td>Bruker</td><td>8237001</td><td>For preparation and section of tissue.</td></tr><tr><td>Coplin Jars</td><td>Sigma Aldrich</td><td>S5516</td><td>For preparation and section of tissue.</td></tr><tr><td>Pressure Cooker</td><td>Kambrook</td><td>KPR620BSS</td><td>For preparation and section of tissue.</td></tr><tr><td>Sublimator</td><td>Chem Glass</td><td>NA</td><td>For sublimation procedure. Similar in design to the CG-3038 however it was custom made&amp;nbsp;</td></tr><tr><td>Sand bath</td><td>NA</td><td>NA</td><td>For sublimation procedure. Fine grade river sand held in folded aluminium foil sourced from outside not from any specific company</td></tr><tr><td>Glass Petri Dish</td><td>Sigma Aldrich</td><td>CLS70165100</td><td>For sublimation procedure.</td></tr><tr><td>Vacuum Pump</td><td>NA</td><td>NA</td><td>For sublimation procedure. Sourced as a spare part from an old mass spectrometer&amp;nbsp;</td></tr><tr><td>Cold trap</td><td>Chem Glass</td><td>CG-4510-02</td><td>For sublimation procedure.</td></tr><tr><td>Hot Plate</td><td>John Morris</td><td>EW-15956-32.&amp;nbsp;</td><td>For sublimation procedure.</td></tr><tr><td>Plastic petri dish</td><td>Sigma Aldrich</td><td>Z717223</td><td>For sublimation procedure.</td></tr><tr><td>37 &amp;deg;C incubator</td><td>NA</td><td>NA</td><td>For sublimation procedure. Not applicable, incubator is non sterile and over 30 years old&amp;nbsp;</td></tr><tr><td>Blotting paper</td><td>Sigma Aldrich</td><td>P7796</td><td>For sublimation procedure.</td></tr><tr><td>Nitrocellulose&amp;nbsp;</td><td>Sigma Aldrich</td><td>N8395</td><td>For washing of slides.</td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td>650501</td><td>For washing of slides.</td></tr><tr><td>Xylene</td><td>Sigma Aldrich</td><td>214736</td><td>For washing of slides.</td></tr><tr><td>100% EtOH</td><td>Sigma Aldrich</td><td>1.02428</td><td>For washing of slides.</td></tr><tr><td>70% EtOH</td><td>Sigma Aldrich</td><td>NA</td><td>For washing of slides. Made in lab from 95% stock ethanol&amp;nbsp;</td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>C2432</td><td>For washing of slides.</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma Aldrich</td><td>ARK2183</td><td>For washing of slides.</td></tr><tr><td>Tris HCL pH 8.8</td><td>Sigma Aldrich</td><td>TRIS-RO</td><td>For proteolytic cleavage. Powder made to 1M followed by equilibration with 32% HCl to PH 8.8</td></tr><tr><td>Milli Q Ultra-Pure Water</td><td>Sigma Aldrich</td><td>NA</td><td>For proteolytic cleavage. Purification performed in house by sartorious water purification system</td></tr><tr><td>Ammonium Bircarbonate</td><td>Sigma Aldrich</td><td>A6141</td><td>For proteolytic cleavage.&amp;nbsp;</td></tr><tr><td>Trypsin</td><td>Sigma Aldrich</td><td>T0303</td><td>For proteolytic cleavage.&amp;nbsp;</td></tr><tr><td>CHCA Matrix</td><td>Sigma Aldrich</td><td>C2020</td><td>For recrystallisation.</td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>1.00029</td><td>For recrystallisation.</td></tr><tr><td>Trifluoroacetic Acid (TFA)&amp;nbsp;</td><td>Sigma Aldrich</td><td>302031</td><td>For recrystallisation.</td></tr></tbody></table>

optimal preparation of formalin fixed samples for peptide based matrix assisted laser desorption/ionization mass spectrometry imaging workflows

基质辅助激光解吸/电离, 质谱成像 (MALDI MSI) 的使用迅速扩大, 因为这项技术分析了大量的生物分子从药物和血脂到 n-糖。尽管存在各种样品制备技术, 但从甲醛保存的组织中检测多肽仍然是这类质谱分析中最困难的挑战之一。基于这个原因, 我们创建并优化了一个健壮的方法, 它保留了样本中包含的空间信息, 同时引出了最大数量的电离肽。我们还旨在以成本效益和简单的方式实现这一目标, 从而消除在使用自动化检测时可能出现的潜在偏差或准备错误。最终结果是一个可重现且价格低廉的协议。

如果正确遵循, 该协议将生成清晰地表示组织的大体形态的图像, 而不需要任何划痕或其他变形 (图 1)。正确执行样本准备的理想验证是通过改变被查看的分子 (图 2) 区分不同物理结构的能力。
一个很好的指南, 确定样本是否已不正确的准备是检查是否存在域或矩阵聚合;延伸到物理?...

Watch this Scientific Journal Video about Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows at JoVE.com

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

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

本协议描述了一种可重现且可靠的方法, 用于 sublimation-based 制备用于成像质谱的福尔马林固定组织。

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

optimal-preparation-formalin-fixed-samples-for-peptide-based-matrix

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.0K 次观看.  University of Sydney. 本协议描述了一种可重现且可靠的方法, 用于 sublimation-based 制备用于成像质谱的福尔马林固定组织。

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

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

定量应用同质MALDI样品的制备

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

科研

生物化学

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 ...

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

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

生物工程

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

使用胺还原甲基化进行样品制备和相对定量，用于肽学研究

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

免疫与感染

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

生物学

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

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

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

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

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

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

组织样品质谱基猎枪蛋白造影学的综合工作流程

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

使用 MALDI 质谱成像进行代谢分析的示例准备

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...