叶贺和里纳特·阿布扎利莫夫得到了纽约城市大学专业工作人员大会（PSC-CUNY）研究奖项目的支持。陈玉琪和凯利·维拉萨米得到了阿尔弗雷德·斯隆基金会CUNY暑期本科生研究计划的支持。

该协议遵循纽约城市大学高级科学研究中心（ASRC）机构动物护理和使用委员会（IACUC）的指导方针。
1. 收获组织
<ol><li>准备铝船。准备一个长方形铝10厘米×20厘米，折叠在中间，使10厘米双层方形。将样品信息标记在一边，将另一侧折叠起来，形成一条底部表面约 4 厘米 x 4 厘米的船。</li>
	<li>在泡沫泡沫泡沫盒中的液氮（LN2）上预制船。 注：如果船太小，样品可能会从船上掉下来，并通过直接接触LN2而变得过度冻结，这可能导致在冷冻过程中出现碎片。</li>
	<li>按照国际兽联的制度指南，通过宫颈错位对动物实施安乐死，立即解剖感兴趣的组织。<ol>
<li>尽可能缩短动物麻醉和捕捉冻结之间的间隔，以尽量减少组织采集过程中代谢物的改变，特别是死后缺血导致大脑中的实验室代谢物。 注：用磷酸盐缓冲盐水（PBS）灌注动物将增加缺血的持续时间，进一步改变阴唇代谢物的浓度，夸大人工结果。因此，不建议用 PBS 灌注或清洗组织，除非血液或体液的污染比单个项目的代谢物变质或洗涤更令人关注。</li>
	</ol>
</li>
	<li>立即将组织放入漂浮在液氮上的铝船上，关闭发泡胶盒的盖子，根据组织大小冷冻2-10分钟：产后第1天（P1）、P21、P60鼠脑分别冻结2分钟、5分钟、7分钟，P60大鼠大脑分别冻结10分钟。 注意：不要长时间冻结组织（例如P1小鼠大脑5分钟），因为过度冷冻可能导致冷冻过程中组织碎片。</li>
	<li>用钳子取出船，折叠铝箔包裹纸巾，在干冰上运输，储存在-80°C，供以后使用。如果随后立即进行分割，将干冰上的样品带到低温状态。 注：为了更好地保存代谢物，最好将样品储存在完整的组织中，并在进行 MALDI 成像之前将其分割。组织可在 -80 °C 中存储长达 24 个月。</li>
</ol>2. 组织冷冻
注意：在处理氧化钛 （ITO） 幻灯片时，请随时戴上手套。避免直接呼吸到滑梯上或戴口罩（可选），以防止人唾液污染组织部分。
<ol><li>在分割组织之前，收集与 MALDI 兼容的 ITO 涂层玻璃滑梯所需的数量。</li>
	<li>使用设置为电阻的伏特计测试幻灯片的导电性。标记电阻测量读取的侧面：这将是组织部分所坚持的侧面。务必将滑梯放在干净的纸巾上以避免污染。</li>
	<li>在设置为 -20 °C 的低温统计中预冷却幻灯片。</li>
	<li>如果从 -80 °C 中取出组织样本，则根据组织大小，让组织在低温室中均等约 45-60 分钟。如果样品从干冰中取出，则将其平衡约30分钟。</li>
	<li>用70%乙醇彻底清洁低温剂。预冷所有必要的工具，包括薄尖艺术家刷和钳子在低温室。</li>
	<li>根据组织类型设置低温室和标本头的温度：肝脏 -14 °C，肌肉 -20 °C，皮肤9-25 °C。</li>
	<li>使用冷冻组织嵌入化合物 OCT 将组织安装到夹头上，避免 OCT 从感兴趣的区域。</li>
	<li>将干净的刀片放在舞台和锁上。调整舞台位置和标本角度，实现所需的切割角度。 注意：如果要切割不同类型的组织/基因型，请务必重新定位样本，以便使用刀片的清洁部分，或在切割下一个样本之前更改为新的刀片，以防止交叉污染。</li>
	<li>继续切割，直到发现感兴趣的区域（例如，大脑中的语料库）。请务必通过用在低温统计中平衡的艺术家画笔刷掉额外的作品来保持舞台清洁。</li>
	<li>一旦所需的区域被揭示出来，削减10-12μm厚度的较小部分。如果该部分容易剥落或脱落，则将低温统计器的温度提高，保持在 -22 °C 至 -11 °C 范围内。我们发现脑组织的最佳切割温度是 -15 °C 至 - 18 °C。</li>
	<li>一旦一个好的部分被切断，坚持它到ITO幻灯片（在低温室操作）。</li>
	<li>使用艺术家画笔尖将组织的一部分转移到 ITO 幻灯片上。</li>
	<li>将手指放在滑梯下加热部分，以确保安装安全。组织部分将首先在5-10s中变成透明，然后在大约30-60s中变成不透明。</li>
	<li>小心地将幻灯片放在低温统计器中。</li>
	<li>重复其他组织样本的步骤，确保组织的每一部分均匀地放置在幻灯片上，并尽可能对齐。</li>
	<li>由于 MALDI 目标最多可容纳两个幻灯片，请将来自同一组的多个样本的部分放置在单个幻灯片或两个幻灯片上，以确保可以同时分析它们。</li>
	<li>完成后，将 ITO 滑动放入真空箱中，并转移到带干燥剂的干燥器中。真空干燥滑梯 45-60 分钟。 注意：如果实验室中没有真空干燥器，请一直将滑梯保持在 -20 °C 以下，以避免代谢物变质。</li>
	<li>幻灯片存储和运输：除非样本由 MALDI 成像核心设施准备，以便直接进行 MALDI 成像，否则将幻灯片存储在 -80 °C 或运送到干冰上的核心设施或其他 MALDI 研究实验室。</li>
	<li>为了最好地保存样品，将滑梯放入滑梯运输机中，用氮气（可选）填充，用准胶片密封，放入拉链袋中，然后放入另一个装有干燥剂的拉链袋中。给外拉链袋贴上标签。</li>
	<li>在 -80 °C（最多 6 个月）下继续储存或用足够的干冰运输。</li>
</ol>3. 矩阵准备
<ol><li>准备矩阵。 注：所有试剂必须是 HPLC 等级。<ol>
<li>以 10 毫克/mL 的浓度准备 NEDC。准备10 mL的基质溶解在溶剂70%甲醇（100毫克NEDC，7mL甲醇，3mL的H2O）。</li>
		<li>此外，还要准备额外的 10 mL 70% 甲醇：水溶液，以便在填充矩阵之前冲洗喷雾器系统。</li>
	</ol>
</li>
	<li>一旦幻灯片在步骤 2.17 中脱水，使用大胆的点银标记在组织部分外的玻璃滑梯空白处放置"X"标记，然后在银"X"顶部放置带有细点黑色标记的第二个"x"。黑色"X"与银色背景形成鲜明对比（大胆的银色"X"）稍后将作为 MALDI 仪器中后期质量光谱采集的受托标记。</li>
	<li>将滑梯加载到 MALDI 滑动金属目标中。使用塑料盖，将样品绘制到塑料盖上。留。</li>
	<li>使用平板扫描仪与 MALDI 目标一起扫描幻灯片图像。目标表面的螺丝将用作垫片，以防止扫描仪损坏或污染组织部分。在 16 位灰度和 2400 dpi 中预览和扫描选定的幻灯片区域。保存图像，以便以后在马尔迪 MSI 中使用。</li>
</ol>4. 矩阵沉积
注：有多种方法将细晶体大小的均匀矩阵层应用于 MALDI 幻灯片，包括升华、滴喷墨打印、自动矩阵喷雾器和使用艺术家 airbush9的手动喷雾。我们将使用自动矩阵喷雾器作为本协议中高可重复性示例。
<ol><li>启动：打开矩阵喷雾器单元，确保阀门位于 LOAD 位置并启动喷雾器软件。检查排气风扇是否运行良好。如果活性通风不能正常工作，请不要启动溶剂泵。</li>
	<li>在 Comms 选项卡上确认一切都在通信，然后以 0.1 mL/min 启动溶剂泵，背压为 ±500 psi（或 3.4 MPa）。</li>
	<li>通过将氮气罐设置为 30 psi 来开始压缩流向矩阵喷雾器的气流。然后，将喷雾器前部的压力调节器调整为 10 psi，并根据需要设置喷雾喷嘴温度。 注意：如果祈祷中的气压低于 5 psi，喷雾喷嘴将无法加热以保护。</li>
	<li>由于阀仍在 负载 位置，请使用注射器用 7 mL 的 70% 甲醇冲洗环路，然后用 6 mL 矩阵填充环。</li>
	<li>将组织滑入喷雾器中的支架，将两端贴上胶带以防止移动，并保留无基质边缘，以避免矩阵对 MALDI 目标的金属夹子进行污染。 注：强烈建议在进入珍贵样品幻灯片之前，先在空白显微镜幻灯片上测试矩阵喷雾。</li>
	<li>检查流量和温度是否稳定，开始喷洒。</li>
	<li>使用空白玻璃滑梯选择所需的方法进行预测试。</li>
	<li>按 开始。这将设置喷嘴温度并调整泵流量，以匹配所选方法。将阀门从 负载 切换到 喷雾 位置，然后通过单击 "继续"进行确认。</li>
	<li>允许系统运行，根据方法，每个幻灯片需要 5-20 分钟。将阀门从 喷雾切换 到 负载 位置，然后在完成后单击 "继续" 进行确认。</li>
	<li>从喷雾器中取出幻灯片，检查显微镜下矩阵涂层的图案，以确保细矩阵晶体的均匀层。</li>
	<li>矩阵沉积后，将幻灯片放入金属 MALDI 支架中立即使用。如果只有一个标本幻灯片，则添加另一个空白幻灯片以填充 MALDI 支架的两个空格。</li>
	<li>按照制造商的指示在使用后立即清理系统，以防止喷雾喷嘴堵塞。</li>
	<li>样品幻灯片涂上矩阵后，要么立即进入 MALDI 成像仪，要么使用第 2.19 步描述的相同双拉链袋制备，用干冰将其装运。 注：在紧急情况下，如 MALDI 仪器不可立即使用，将涂层滑梯存放在真空状态或 -20 °C 下长达 24 小时，尽管某些代谢物的恶化可能发生在存储期间，但尚未彻底研究。</li>
</ol>

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作者声明没有相互竞争的经济利益。

MALDI-成像（MALDI-MSI）是一种无标签的成像技术，使研究人员能够研究各种生物分子的分布及其在组织中的修饰，这是病理学的分子基础。将 MALDI-MSI 与传统的 LC-MS 组织分析方法相结合，提供与传统 Omics 工作流相同的分子深度，但也保留了细胞网络中这些信号的空间关系。样本制备是 MALDI MSI 中最关键的一步，也是在不同实验室进行的代谢学研究的最终读出中变化的原因。在这里，我们提供了一个全面而实用的协议，以标准化使用 MALDI MSI 进行代谢分析的样本准备，希望它有利于广泛的研究团体在其当前和未来的研究中实施 MALDI MSI，从基础生物学到转化研究。
人们必须始终牢记所有预防措施，以尽量减少样品制备过程中分子剖面（丰度和空间分布）的变化，避免污染。首先，尽量减少动物安乐死和组织采集之间的时间，如原地冷冻或微波固定加热，使大脑中的酶失活，以减少对死后缺血症的责任4，5，6。其次，样品的捕捉冻结状态至关重要。冷冻不足会导致代谢物的退化和损失，而过度冷冻会导致冷冻过程中的组织碎片。冷冻时间应始终根据先前报告的研究进行测试，本文中对发育小鼠大脑的研究为啮齿动物脑组织提供了参考点。第三，切割生物组织并将其部分转移到 ITO 幻灯片中需要充分实践。请务必注意，在使用画笔从舞台上拾取部分时，应小心使用画笔。允许刷毛只接触组织部分的边缘，以降低污染和部分碎裂的风险。尽可能平整滑梯上的部分，这样可以防止在手指加热期间卷曲该部分。第四，在安装到 ITO 幻灯片上时，请确保整个部分与 ITO 幻灯片连接良好，因为组织的不同区域可能需要不同时间的手指加热。例如，脑肿瘤组织需要比正常脑组织更长的加热时间。在 MALDI-MS 扫描过程中，安装不良可能导致组织分离和碎裂。请记住，手指变暖可能使酶作用和新陈代谢导致代谢的人工变化。第五，MALDI矩阵的精细和均匀沉积在实现准确的空间信息和强 MALDI-MS 信号方面发挥了重要作用。建议在空白幻灯片上测试矩阵喷洒，并在显微镜下观察晶体图案以验证适当的覆盖范围，然后再进行珍贵标本幻灯片的涂层。最后，由于单个研究人员以不同速度执行组织采集和滑动制备，因此最好由一个人在同一组中处理样本制备，以最大限度地减少变异。
协议提供了上述详细标准程序，可根据特定实验的需要进行定制。例如，通常用于样品冷冻分割的冷冻凝胶 OCT 可以进一步用作组织夹头的安装胶（如上文所述）。先前的研究表明，OCT中的聚合物成分会导致强烈的离子抑制14。然而，在没有聚合物凝胶的额外支持的情况下，嵌入样品可能是不可避免的。为了对抗这些情况下的信号抑制，组织可能需要用70%乙醇和95%乙醇的连续清洗清洗，以去除残留的OCT来检测蛋白质或脂质，而不建议清洗用于检测小分子代谢物9。
MALDI MSI 在研究实验室和临床实践环境中越来越相关。例如，MALDI MSI最近被证明在研究蛋白造物学，以描述一个有机体15的表型功能状态，并作为微生物识别和诊断随后疾病16的代理。虽然 MALDI MSI 支持广泛的应用，但仅依靠此技术存在一些限制，尤其是在区分相似物种或代谢物以及确定特定目标方面。另一个挑战是根据 MALDI MSI 信号对代谢物浓度进行量化。人们通常认为，MALDI MSI光谱中的离子丰度与解剖组织中相应分子物种的空间分布（或相对丰度）密切相关。然而，人们应该始终记住，离子强度与相应分子物种数量之间的关系是由许多因素造成的，包括但不限于离子抑制的影响、组织结构的变化和离子分子反应17。利用内部标准的技术可以实施在MALDI-MSI18中进行绝对量化（μmol/g组织）。这两个挑战通常通过 MALDI MSI 与液相色谱串联 MS （LC-MS/MS） 技术相结合的工作流程来解决，根据该技术，MALDI-MS 允许绘制感兴趣的区域图谱，该区域随后需要微切除和 LC-MS/MS 来提供更多信息来识别代谢物19。
近年来，基于MS的成像方法被开发出来，作为以前对小分子代谢物进行成像技术的替代方式。随着 MALDI MSI 的进步和日益普及，预计 MALDI 成像将成为可视化小分子的新标准工具。在生物环境中对脂质和内源性小分子（如神经递质和代谢物）进行成像，以及为开发新药剂而成像异生物，都特别令人感兴趣。预计在不久的20年，随着马尔迪MSI的应用，这三个领域将取得迅速进展。

代谢物，代谢的中间体或最终产物，包括核苷酸、氨基酸或有机酸、脂质，是生物功能和过程的关键成分。代谢学，代谢物的研究，允许探索其生化相互作用，并理解其在基础、转化和临床研究中的作用。代谢物与生物体的表型密切相关，并提供细胞代谢过程中发生的生化活动信息。因此，除了基因组学和益生术之外，代谢学已成为了解生理和病理条件的重要工具。例如，代谢学用于阐明现有药物背后的机制及其耐受性。在药物开发中，异生物代谢有助于评估不同物种代谢物的活动或毒性，后来转化为支持个性化药物2。尽管代谢学的广泛应用，代谢物的成像可能具有挑战性，由于代谢物的化学反应性，结构异质性和广泛的浓度范围3。然而，包括高能化合物、葡萄糖、乳酸盐、糖溶胶、五氯蔗分流通路和TCA循环中间体、磷脂、神经递质、信号化合物在内的阴唇代谢物的浓度，在组织酶在组织采集过程中活跃时，如脑收获4、5、6中的死后缺血，可在几秒钟内发生变化，并在几分钟内取得进展.为确保准确的代谢数据采集，适当和仔细的样品准备是关键7，8。目前已建立的测量代谢物的平台包括NMR、酶检测和质谱（包括液体和气体色谱），最后一个平台在下面进一步讨论。
MALDI-MSI 是一种最先进的技术，它允许通过检测单个分子物种来分析复杂的样品。MALDI MSI 使能够快速和可重复地测量生物样本中的各种分子化合物的好处。质谱成像进一步允许产生基于其复合代谢物的表示组织生物学的图像，同时保留样品9中代谢物的空间分布。MALDI 能够在不使用抗体标记、结构修饰或其他专用试剂（如用于免疫染色）的情况下检测样品中的分析物，再加上它在单个实验中监测数百个分子的能力，仅包括 MS 成像在代谢分析10方面授予的一些优势， 11.除了常用的基质，如2，5-二氢苯二甲酸（DHB）和9-阿米诺阿克里丁（9-AA），最近发现了新的基质N-（1-纳芙yl）二氢二氢化二氢（NEDC），这是非常适合分析各种低分子量代谢物， 进一步改善了马尔迪MSI在代谢分析12中的应用。
尽管MALDI MSI的广泛应用，但仪器的高成本和实验程序的复杂性阻碍了其在个别研究实验室的广泛实施。因此，大多数 MALDI MSI 研究都通过共享的核心设施提供支持。样品制备，包括幻灯片制备和矩阵涂层，是 MALDI MSI 中最关键的一步。但是，幻灯片制备通常在个别研究人员的实验室进行，这为以后的 MALDI MSI 收购带来了潜在的变化。在这里，我们旨在提供一个详细的协议，为生物样本的样品准备之前，进行MALDI MSI测量，并使用发育小鼠大脑的代谢分析作为例子。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Andwin Scientific&#160;Tissue-Tek CRYO-OCT Compound</td>
			<td>Fisher Scientific&#160;</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Artist brush MSC #5 1/8 X 9/16 TRIM RED SABLE</td>
			<td>Fisher Scientific&#160;</td>
			<td>50-111-2302</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoflex speed MALDI-TOF MS system</td>
			<td>Bruker Daltonics Inc</td>
			<td></td>
			<td>MALDI-TOF MS instrument</td>
		</tr>
		<tr>
			<td>BD&#160;Syringe with Luer-Lok Tips</td>
			<td>Fisher Scientific&#160;</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Vacutainer General Use Syringe Needles</td>
			<td>Fisher Scientific&#160;</td>
			<td>23-021-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Daltonics&#160;GLASS SLIDES MALDI IMAGNG</td>
			<td>Fisher Scientific&#160;</td>
			<td>NC0380464</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite, with indicator, 8 mesh, ACROS Organics&#160;</td>
			<td></td>
			<td>AC219095000</td>
			<td></td>
		</tr>
		<tr>
			<td>Epson Perfection V600 Photo Scanner</td>
			<td>Amazon</td>
			<td>Perfection V600</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;5-Place Slide Mailer</td>
			<td>Fisher Scientific&#160;</td>
			<td>HS15986</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Digital Auto-Range Multimeter</td>
			<td>Fisher Scientific&#160;</td>
			<td>01-241-1</td>
			<td></td>
		</tr>
		<tr>
			<td>FlexImaging v3.0</td>
			<td>Bruker Daltonics Inc</td>
			<td></td>
			<td>Bruker MS imaging analysis software</td>
		</tr>
		<tr>
			<td>HPLC Grade Methanol</td>
			<td>Fisher Scientific&#160;</td>
			<td>MMX04751</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Grade Water</td>
			<td>Fisher Scientific&#160;</td>
			<td>W5-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HTX M5 Sprayer</td>
			<td>HTX Technologies, LLC</td>
			<td></td>
			<td>Automatic heated matrix sprayer</td>
		</tr>
		<tr>
			<td>Kimberly-Clark Professional&#160;Kimtech Science Kimwipes Delicate Task Wipers</td>
			<td>Fisher Scientific&#160;</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>MSC&#160;Ziploc Freezer Bag</td>
			<td>Fisher Scientific&#160;</td>
			<td>50-111-3769</td>
			<td></td>
		</tr>
		<tr>
			<td>N -(1-Naphthyl) Ethylenediamine Dihydrochloride (NEDC)</td>
			<td>Millipore Sigma Aldrich</td>
			<td>222488</td>
			<td></td>
		</tr>
		<tr>
			<td>SCiLS Lab (2015b)</td>
			<td>SCiLS Lab</td>
			<td></td>
			<td>Advanced MALDI MSI data analysis software</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;CryoStar NX50 Cryostat</td>
			<td>Fisher Thermo Scientific</td>
			<td>95-713-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Nalgene Transparent Polycarbonate Classic Design Desiccator</td>
			<td>Fisher Scientific&#160;</td>
			<td>08-642-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation for metabolic profiling using maldi mass spectrometry imaging

代谢学是识别和量化实验样本中小分子和代谢物的研究，已成为研究发育过程中的生物活动和疾病的重要工具。代谢学方法被广泛应用于癌症、营养/饮食、糖尿病和其他涉及代谢过程的生理和病理条件的研究。本文所提倡的有助于代谢分析的有利工具是矩阵辅助激光脱光/电离子质谱成像（MALDI MSI）。它能够在不标记、结构修改或其他特殊试剂（如用于免疫染色）的情况下就地检测代谢物，使 MALDI MSI 成为推进代谢学领域相关方法的独特工具。适当的样品制备过程对于产生最佳结果至关重要，并且将成为本文的重点。

具有代表性的实验是根据 图1中显示的工作流程进行的。产后第1天、第21天、第60天（成人）发育的C57BL野型小鼠大脑按照CUNY IACUC指南采集，分别在漂浮在液氮上的铝船上冷冻2、5和7分钟。冷冻组织在标本头和腔室的 +15 °C 厚度段下冷冻。然后，组织低温被轻轻地转移到 ITO 涂层玻璃幻灯片的预冷却导电侧，以便进行 MALDI 成像。在室温下，ITO 幻灯片上的安装低温在真空中干燥 45 分钟，然后使用自动矩阵喷雾器进行矩阵沉积。矩阵 NEDC 用于检测代谢物，甲醇/水中（70/30 v/v）中 10 毫克/mL 的矩阵溶液以 0.1 mL/min 的流速沉积，喷嘴温度为 75 °C，用于 12 个周期，每个周期之间有 5 s 干燥。采用喷雾速度1300毫米/分钟，轨道间距2毫米，N2 气压10 psi，流速3L/min，喷嘴高度40毫米。
MALDI质量光谱由马尔迪飞行时间（TOF）MSI仪器以负离子模式获得。0.5-1 μL 的红磷（Pn 簇与 n = 1 - 90）甲醇乳液沉积到 ITO 幻灯片上，旁边安装的组织，并用于校准仪器在 100 -1000 m/z 质量范围内应用二次校准曲线13。激光点直径聚焦于 50μm 光刺宽度的"中等"调制光束轮廓。质量范围从 m/z 50 到 1000 的光谱以 1000 Hz 获得，用于 500 次拍摄。记录了质谱数据，并使用先进的 MALDI MSI 数据分析软件进一步分析了成像。离子图像生成时，根均值方块 （RMS） 正常化的垃圾箱宽度为 ±0.25 Da。
图2的结果显示，从每100道尔顿间隔中选择的M/z光谱MALDI MSI数据分析软件的输出图像，清楚地描绘了从小分子代谢物到高分子量脂质的光谱识别效用。每行都绘制各自的离子热图，包含产后第 1、21 和 60 天收集的三个组织中特定代谢物物种的空间和光谱信息。对于每个具有代表性的 m/z，可以使用区域分布和离子丰度分析来比较不同年龄的相应物种的相对数量。MALDI MSI 方法的一个优点是能够识别 m/z 识别的某些物种对发育里程碑或特定解剖结构的特异性。观察到一些代谢物在P1新生儿（m/z 320.1）中富集，在P60成人中富集（m/z 846.5），或在不同年龄（m/z 480.3）中均匀分布：其他分子物种特别富含灰质（m/z 117.1：524.3：765.1）、白质（m/z 673.4：846.5）或CSF/文特尔（m/z 239.0）（图2A）。代表性代谢物的空间分布，包括低氧酸（m/z 135.0）、N-乙酰-L-阿斯巴酸（m/z 174.0）、阿拉奇多尼奇 酸 （m/z 303.2）， 和几个脂质， 如利索法蒂莱他诺胺 LPE （18：1） （m/z 478.3）， LPE （20：4） （m/z 500.3）， LPE （22：6） （m/z 524.3）， 磷酸乙醇胺PE （44：： 10） （m/z 838.5），磷酸二醇PI（38：4）（m/z 885.6）也显示（图2B）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62008/62008fig01.jpg"> 图1。马尔迪-飞行时间（TOF）质谱成像的工作流程。 捕捉冷冻组织在低温统计中低温，并安装在 ITO 幻灯片上。→ 带组织部分的幻灯片使用自动矩阵喷雾器涂上细细的矩阵层。→质谱由马尔迪-托夫MSI仪器在20-200um的光泽中收集。→ 数据进行分析，并且使用先进的 MALDI MSI 数据分析软件生成图像。缩放栏：2 毫米。 <a href="https://www.jove.com/files/ftp_upload/62008/62008fig01large.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/62008/62008fig2v2.jpg"> 图2。代表输出，选定的 m/z 光谱来自质谱学，以 50 μm 的横向分辨率获得。（A） 热图描绘了从P1、P21和P60确定的三个发育里程碑中每100道尔顿/z间隔中选择的特定代谢物物种的空间分布。（B） P1、P21和P60中代表性代谢物的空间分布，包括：低氧酸、N-乙酰-L-阿斯巴酸、阿拉奇多尼酸和不同脂质物种，如利索法蒂莱诺胺（LPE）、磷酸二醇胺（PE）、磷脂二醇（PI）。缩放栏，500 μm。<a href="https://www.jove.com/files/ftp_upload/62008/62008fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging at JoVE.com

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

本协议的目标是在规划使用 MALDI MSI 进行实验时，为样品制备提供详细的指导，以最大限度地提高生物样品中的代谢和分子检测。

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 ...

sample-preparation-for-metabolic-profiling-using-maldi-mass

Research

JoVE Journal

Biology

5.9K 次观看.  The City University of New York. 本协议的目标是在规划使用 MALDI MSI 进行实验时，为样品制备提供详细的指导，以最大限度地提高生物样品中的代谢和分子检测。 

视频: Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

酰胺氢/氘交换和MALDI - TOF质谱PAK2激活的质谱分析

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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

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

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

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

MALDI-质谱成像代谢物的研究<em&gt;苜蓿</em&gt;根瘤

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

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...