A Highly Multiplexed, Multiomic 3D Mouse Brain Map Using MALDI-IHC

摘要/摘要 NIH脑计划的一个中心目标是开发新的成像工具，这些工具足够强大，可以在空间上映射 高分辨率的神经元回路和大脑的基本分子组成。当尖端 尽管已经开发了成像工具和相关的标记技术，但绘制空间分布图仍然是一个重大挑战。 分布在不同长度尺度的数千个生物分子，包括表达的蛋白质，发挥作用， 在大脑功能中的关键作用。这个第二阶段项目的目标是评估一种新的组织成像的能力， AmberGen开发的称为MALDI-IHC的技术，用于快速创建高度多重化、多组学和 小鼠大脑的多模式3D分子图。全脑成像MALDI-IHC的发展将 为神经科学家探索大脑功能的潜在分子基础提供了重要的新工具 和神经退行性疾病。MALDI-IHC基于使用新型光裂解质量标签（PC-MT） 由AmberGen开发，当与抗体或凝集素探针连接时， 在质谱图像中识别。这种方法大大超过了多路复用能力， 荧光免疫组织化学（IHC）和先前的基于可裂解质量标签的方法，其通常 仅限于5种生物标志物或需要大量的循环程序。它也超过了金属标记的能力 抗体技术，如IMC和MIBI，可以以亚细胞分辨率探测小的mm2区域，但 仅限于大约40个抗体探针并且需要几天来扫描整个组织切片。与此相反， MALDI-IHC可以以40 µ m的分辨率对整个小鼠脑FFPE切片中的100多种靶蛋白进行成像， 不到一个小时。MALDI-IHC进行无标记、非靶向小分子质谱分析的能力 MSI成像、使用独特的双标记荧光-PC-MT探针的荧光成像和高重成像 包括糖基化模式在内的完整表达蛋白质在同一组织切片上的表达大大扩展了 这种方法。在第一阶段，我们证明了这种联合方法在小鼠脑FF上的可行性。 和FFPE组织标本。在第二阶段，我们将开发使用MALDI-IHC重建整个 40 µ m体素分辨率的小鼠脑蛋白表达图。FFPE矢状面和冠状面小鼠脑组织切片 将使用一组50个NeuroMab PC-MT抗体和25个PC-MT抗体通过MALDI-IHC探测来自小鼠脑的 凝集素。将通过比较MALDI-IHC和荧光IHC对单个PC-MT探针进行验证 图像.还将绘制小鼠大脑的3D三模态图，其合并了代谢物和表达的蛋白质。 基于所展示的工作流程进行重建，该工作流程涉及来自连续的未标记的小分子的MSI。 FF标本，使用75重PC-MT探针组（包括一些双标记的PC-MT抗体）进行IHC染色， 和荧光成像，随后进行PC-MT的MSI。3D标测图、可视化和图像重建 将使用Bruker SCiLS ™软件进行分析。MALDI-IHC技术的商业化将是 通过与MALDI-MSI仪器的市场领导者Bruker Daltonics的密切合作，加速了这一进程。

Summary/Abstract A central goal of the NIH Brain Initiative is to develop new imaging tools sufficiently powerful to spatially map at high resolution the neuronal circuitry and underlying molecular composition of the brain. While cutting edge imaging tools and related labeling techniques have been developed, it is still a major challenge to map the spatial distribution at different length scales of the thousands of biomolecules, including expressed proteins, which play key roles in brain function. The goal of this Phase II project is to evaluate the ability of a new tissue imaging technology developed by AmberGen, termed MALDI-IHC, to rapidly create a highly multiplexed, multiomic and multimodal 3D molecular map of the mouse brain. The development of MALDI-IHC for whole brain imaging will provide neuroscientists with an important new tool for exploring the underlying molecular basis of brain function and neurodegenerative disorders. MALDI-IHC is based on the use of novel photocleavable mass-tags (PC-MTs) developed by AmberGen which when linked to antibody or lectin probes enable targeted biomolecules to be identified in the mass spectrometric image. This approach significantly exceeds the multiplex capability of fluorescence immunohistochemistry (IHC) and previous cleavable mass-tag based methods which are generally limited to 5 biomarkers or require extensive cycling procedures. It also exceeds the capability of metal-tagged antibody techniques such as IMC and MIBI which can probe small mm2 regions at subcellular resolution but are limited to approximately 40 antibody probes and require several days to scan a whole tissue section. In contrast, MALDI-IHC can image an entire mouse brain FFPE section for over 100 targeted proteins at 40 µm resolution in less than one hour. The ability of MALDI-IHC to perform label-free, untargeted small molecule mass spectrometric imaging (MSI), fluorescence imaging using unique dual-labeled fluorescent-PC-MT probes and high-plex imaging of intact expressed proteins including glycosylation patterns on the same tissue section greatly extends the power of this approach. During Phase I, we demonstrated the feasibility of this combined approach on mouse brain FF and FFPE tissue specimens. During Phase II, we will develop methods using MALDI-IHC to reconstruct whole mouse brain protein expression maps at 40 µm voxel resolution. FFPE sagittal and coronal mouse brain tissue slices from mouse brain will be probed by MALDI-IHC using a panel of 50 NeuroMab PC-MT antibodies and 25 PC-MT lectins. Validation of individual PC-MT probes will be performed by comparing MALDI-IHC and fluorescence IHC images. A 3D tri-modal map of the mouse brain merging both metabolites and expressed proteins will also be reconstructed based on a demonstrated workflow that involves MSI of unlabeled small molecules from successive FF specimens, IHC staining with a 75-plex panel of PC-MT probes including some dual-labeled PC-MT antibodies, and fluorescence imaging followed by MSI of the PC-MTs. Reconstruction of 3D maps, visualization and image analysis will be performed using Bruker SCiLS™ software. Commercialization of MALDI-IHC technology will be accelerated by a close collaboration with Bruker Daltonics, the market leader of MALDI-MSI instrumentation.