A biochemical approach towards subcellular, label-free molecular imaging

摘要 基于质谱的空间分析使得能够对广泛的内源性代谢物进行无标记研究。 完整生物和临床样本中的生物分子-脂质、肽/蛋白质、代谢物和聚糖。 这一领域的快速发展为当前空间组学研究做出了巨大贡献。然而，在这方面， 基于MALDI的质谱成像的常规空间分辨率，可以说是最广泛的 今天使用的质谱成像技术，大约是传统质量的几十微米， 光谱仪这种分辨率限制对单细胞水平的样品无标记分析造成了障碍。 或亚细胞水平。 最近发展的扩展显微镜已经看到了相当大的成功，在产生荧光 生物结构的图像超出了光学系统的固有空间分辨率。这种能力具有 导致了超分辨率荧光显微镜在生物和临床实验室中的民主化。 然而，目前的扩展显微镜方案和化学与质谱不兼容 成像或分析。 本提案的主要目的是建立一个样品扩增的生化过程，以推动 将现有质谱成像管道的空间分辨率提高到亚细胞状态，即， 微米及以上。为了实现这一目标，我们将开发一个样品聚合和消化协议 优化的质谱成像，其中内源性脂质和蛋白质被拴系到一个 溶胀聚合物网络，并且随后可以在MALDI质谱仪上分析。我们 实验方法包括（1）开发组织聚合的生化管道， 脂质质谱成像的扩展;（2）将管道扩展到多重脂质和蛋白质 检测和成像。成功完成这项建议将为社会提供一个无标签， 亚细胞分辨率的分子成像模式，而无需修改现有的仪器。

ABSTRACT Mass spectrometry-based spatial analysis has enabled label-free investigation of a broad range of endogenous biomolecules—lipids, peptides/proteins, metabolites, and glycans—in intact biological and clinical specimens. Rapid development in this area has contributed enormously to the current spatial omics research. However, the routine spatial resolution of MALDI-based mass spectrometry imaging, arguably one of the most widely used mass spectrometry imaging techniques today, is about tens of micrometers on a conventional mass spectrometer. This resolution limit imposes a roadblock towards label-free analyses of specimens at single-cell or subcellular level in existing mass spectrometry laboratories. Recent development of expansion microscopy has seen considerable success in generating fluorescence images of biological structures beyond the intrinsic spatial resolution of an optical system. Such capability has led to democratization of super-resolution fluorescence microscopy in biological and clinical laboratories. However, current expansion microscopy protocols and chemistries are not compatible with mass spectrometry imaging or analyses. The main objective of this proposal is to establish a biochemical process of sample expansion to push the spatial resolution of existing mass spectrometry imaging pipelines to the subcellular regime, namely, to a few micrometers and beyond. Towards this goal, we will develop a sample polymerization and digestion protocol optimized for mass spectrometry imaging, in which the endogenous lipids and proteins are tethered to a swelling polymer network and can be subsequently analyzed on a MALDI mass spectrometer. Our experimental approaches include (1) development of a biochemical pipeline of tissue polymerization and expansion for lipid mass spectrometry imaging; (2) extension of the pipeline to multiplexed lipid and protein detection and imaging. Successful completion of this proposal will provide the community with a label-free, subcellular-resolution molecular imaging modality without modifying the existing instrumentation.