Accurate, in-depth mapping of proteins on whole-tissue levels provides comprehensive insights into the spatially-organized regulatory processes/networks in tissues, but is challenging. Here we describe a micro-scaffold assisted spatial proteomics (MASP) strategy, based on spatially-resolved micro-compartmentalization of tissue using a 3D-printed micro-scaffold, capable of mapping thousands of proteins across a whole-tissue slice with excellent quantitative accuracy/precision. The pipeline includes robust tissue micro-compartmentalization with precisely-preserved spatial information, reproducible procurement and preparation of the micro-specimens, followed by sensitive LC-MS analysis and map generation by a MAsP app. The mapping accuracy was validated by comparing the MASP-generated maps of spiked-in peptides and brain-region-specific markers with known patterns, and by correlating the maps of the two protein components of the same heterodimer. The MASP was applied in mapping >5000 cerebral proteins in the mouse brain, encompassing numerous important brain markers, regulators, and transporters, where many of these proteins had not previously been mapped on the whole-tissue level.

在整个组织水平上准确、深入地绘制蛋白质图谱可以全面了解组织中空间组织的调控过程/网络，但具有挑战性。在这里，我们描述了一种微支架辅助空间蛋白质组学 (MASP) 策略，该策略基于使用 3D 打印微支架对组织进行空间分辨微区室化，能够以出色的定量精度绘制整个组织切片上的数千种蛋白质/精确。该管道包括具有精确保存空间信息的强大组织微区室化、可重复的微样本采购和制备，以及随后通过 MAsP 应用程序进行灵敏的 LC-MS 分析和图谱生成。通过将 MASP 生成的掺入肽和大脑区域特异性标记物图谱与已知模式进行比较，并通过关联同一异二聚体的两个蛋白质成分的图谱来验证作图准确性。 MASP 用于绘制小鼠大脑中 >5000 脑蛋白的图谱，其中包括许多重要的大脑标记物、调节因子和转运蛋白，其中许多蛋白质以前从未在全组织水平上进行图谱绘制。