Fig. 1: Overview of the spatially-resolved proteomics workflow.

Tissue is mounted onto a slide compatible with laser capture microdissection (LCM). A general overview is depicted in panel a. Tissue is segmented into a regular grid shape (Annotate), and each element of the grid is isolated by LCM into a well of a 96-well plate (Cut). Proteins from each sample are lysed in RIPA buffer (Lyse) and digested into peptides (Digest) before analysis by LC-MS/MS. The quantitative information for each protein can be mapped back to its location within the gridded tissue and visualised in a topographic protein map, with one map per protein quantified (Proteomic Maps). This workflow was applied to an Atypical Teratoid-Rhabdoid Tumour (AT/RT). b A H&E-stained section was segmented into a 24×16 grid and analysed with the workflow to generate a Protein Topography Stack containing over 5,000 proteomic maps at 833 µm resolution, allowing for the resolution of several features within the tissue while maintaining good throughput. c We then proceeded to apply this workflow at smaller length scales. In total, we applied this workflow over three length scales within serial sections of the AT/RT tumour tissue: 833 µm resolution, covering an entire tumour section; 350 µm resolution, covering part of the boundary between two visibly distinct regions; and 40 µm resolution, covering several blood vessels and their surrounding cells. These data relating to these three length scales are shown in Figs. 2, 3, 4 and 5, respectively. Scale bar represents 833 µm in the bottom, 350 µm in the middle image, and 40 µm in the top image.