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>

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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, 以及确保样品是均匀消化和再结晶。必须实现矩阵的均匀沉积, 因为太少会显示出相对较低的强度和物种多样性, 而且过多的离子会从样品中压制出来, 同时也降低了物种的强度和多样性25。为了实现均匀的基体沉积, 确保均匀的薄层基体晶体的大小, 直接放在悬浮显微镜滑动的脚印下面。如果该层不均匀或太薄, 则矩阵的升华将进行得太慢或将不均匀26。手动应用溶剂和酶的潜在问题归结为用户的个人经验。为了确保可重复的结果, 需要某种程度的 "练习", 并且可能有一些最初的意外错误准备。但是, 如果要注意确保最后的图像符合我们提供的 "好" 样品的性质, 那么任何不适当的样品都不会导致错误的生物学结论。
为了有效地消化在甲醛 (FFPE 或 FFF) 中保存的组织样品, 必须在胰蛋白酶沉积16之前尽可能多地除去许多亚甲基通道。这是很容易实现的加热样品在压力锅在 > 100 ° c, 在短时间内, 没有实际造成气泡, 可以机械地离解样品。组织可以容易地丢失, 当对待用这样方式时, 因此为了克服此, NC 幻灯片被使用。虽然不严重, 但它确实确保了样品的物理完整性时, 受到热, 压力, 和有机溶剂。然后通过将酶应用于防止其活化的状态, 然后使其干燥,即在超纯水中悬浮, 从而实现了对样品的消解。蒸气室然后遵循同样的原理, 如再结晶, 提供足够的湿润环境, 使酶的局部移动, 以遇到和切割蛋白质骨干, 但没有足够的 "湿度", 使产生的肽漂移显着从他们的初始位置26。移直接在幻灯片上不会域任何表面分析, 由于残余交联和一般不的大蛋白质在水中的影响。
尽管我们已经将这一方法应用到多肽上, 但它也可以很容易地应用于对代谢产物、血脂和完整蛋白质的分析工作。有些步骤需要删除或扩充;完整的蛋白质成像不需要任何蛋白水解的步骤, 但基本工作流程的样品切片和安装后, 矩阵升华, 再结晶, 和分析可以适用于几乎任何样本类型。通过广泛的经验调查, 我们制定这项议定书和确保其可靠性和稳健性的意图是建立一种不需要任何修改、使用廉价设备的方法, 并能得到广泛具有不同程度的仪器和生化技术的社区。我们希望, 这将为实验室开辟新的调查渠道, 否则将把这种技术视为过于复杂或昂贵。我们还相信, 我们已经提供了第一个确定的肽 MSI 样品制备方法。在编写本报告时, 大多数方法基本上都是仪器的, 特别强调使用基于机器人的喷涂方法的易用性27,28,29。在可靠的亚甲基水解方法方面, 也很少有关于正确的程序和使用的仪器的猜想。
最后, 我们认为, 上述方法的应用 FFPE 和 FFF 组织, 将导致更大的信心, 在微星的功效分析的肽。

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 border="0" cellpadding="0" cellspacing="0" style="width:820px;" width="820">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryo Microtome</td>
			<td style="width:121px;">Leica</td>
			<td style="width:160px;">CM3050</td>
			<td style="width:275px;">For preparation and section of tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Indium Tin Oxide Microscope slides</td>
			<td style="width:121px;">Bruker</td>
			<td>8237001</td>
			<td>For preparation and section of tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Coplin Jars</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>S5516</td>
			<td>For preparation and section of tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Pressure Cooker</td>
			<td style="width:121px;">Kambrook</td>
			<td>KPR620BSS</td>
			<td>For preparation and section of tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sublimator</td>
			<td style="width:121px;">Chem Glass</td>
			<td>NA</td>
			<td>For sublimation procedure. Similar in design to the CG-3038 however it was custom made&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sand bath</td>
			<td style="width:121px;">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 height="21">
			<td height="21" style="height:21px;width:264px;">Glass Petri Dish</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>CLS70165100</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Vacuum Pump</td>
			<td style="width:121px;">NA</td>
			<td>NA</td>
			<td>For sublimation procedure. Sourced as a spare part from an old mass spectrometer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cold trap</td>
			<td style="width:121px;">Chem Glass</td>
			<td>CG-4510-02</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Hot Plate</td>
			<td style="width:121px;">John Morris</td>
			<td>EW-15956-32.&#160;</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Plastic petri dish</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>Z717223</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">37 &#176;C incubator</td>
			<td style="width:121px;">NA</td>
			<td>NA</td>
			<td>For sublimation procedure. Not applicable, incubator is non sterile and over 30 years old&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Blotting paper</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>P7796</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Nitrocellulose&#160;</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>N8395</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Acetone</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>650501</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Xylene</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>214736</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">100% EtOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>1.02428</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">70% EtOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>NA</td>
			<td>For washing of slides. Made in lab from 95% stock ethanol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Chloroform</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>C2432</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Glacial Acetic Acid</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>ARK2183</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Tris HCL pH 8.8</td>
			<td style="width:121px;">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 height="21">
			<td height="21" style="height:21px;width:264px;">Milli Q Ultra-Pure Water</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>NA</td>
			<td>For proteolytic cleavage. Purification performed in house by sartorious water purification system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Ammonium Bircarbonate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>A6141</td>
			<td>For proteolytic cleavage.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Trypsin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>T0303</td>
			<td>For proteolytic cleavage.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CHCA Matrix</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>C2020</td>
			<td>For recrystallisation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Acetonitrile</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>1.00029</td>
			<td>For recrystallisation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Trifluoroacetic Acid (TFA)&#160;</td>
			<td style="width:121px;">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;延伸到物理组织边界之外的多肽签名是分子在样品制备过程中移动的完美指示器。这在 O 的洛克和 Padula (2017)15中详细说明。
所产生的肽谱应包含高丰度的离散分子很好地分布在选择的质量范围1,23 (这主要是样本依赖, 但是, 它是不寻常的, 看到超过50来自 FFPE 组织的离散肽签名) (图 3)18。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56778/56778fig1.jpg"> 图 1: 正确制备的 FFF 人脑组织标本.该处理后的组织标本已被成像50µm, 显示良好的宏观结构和明显的白色物质 (WM) 和灰质 (GM) 之间的差异。成功制备的样品将显示出不同组织部位的清晰分化。在这里, 有一个明确的区域的多肽 (代表的荷比, M/Z) 1085 在 WM 地区的小组 a. 差异进一步证明, 在小组 B 中没有定义的形状, 然后在小组 C 的返回。通过对同一组织切片上的三种不同分子的分布进行测定, 表明有些是均匀分布的, 而另一些则限于离散的物理位置, 我们可以看到样品的准备工作已经正确地完成了。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56778/56778fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56778/56778fig2.jpg"> 图 2: FFF 人脑组织的一组不正确的准备.这个标本已被成像在50µm, 但显示一个 large-scale 水平的域。1、2和3面板中的分子的模式清楚地表明, 与图 1不同, 在生物区域之间没有明确的定义, 也没有在所显示的分子丰度上的差异。由于图像是相同的, 无论所选择的分子显示, 我们可以得出结论, 它已经域由于不正确的准备。由于这是脑组织, 这一结果并不合乎逻辑, 生物学意义。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56778/56778fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56778/56778fig3.jpg"> 图 3: 显示在中的图像的全局质量频谱图 1。该光谱包含了大量的肽签名横跨低端的质量范围, 并有几个高质量肽峰的存在。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56778/56778fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/56778/56778supfig1.jpg"> 补充图 1: 用于在蒸气室中切割印迹纸的示意图。厚的印迹纸被切成圆形, 直径为94毫米。一个长方形的标签也切出来, 以符合大小的标准玻璃显微镜幻灯片。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56778/56778supfig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/56778/56778supfig2.jpg"> 补充图 2: 蒸汽室的装配示意图。这一示意图显示了如何在蒸气室应该如何组装的具体说明如何的样品幻灯片, 印迹纸和胶带都相互对应。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56778/56778supfig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplementary Figure 3" src="/files/ftp_upload/56778/56778supfig3.jpg"> 补充图 3: 升华室组件示意图:这个数字显示了样品的滑动、升华和加热装置是如何被安排以允许再生升华。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56778/56778supfig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

使用自动工作站进行质谱测量的等离子体样品制备

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.