Abstract
BACKGROUND: Spatial proteomics seeks to understand the spatial organization of proteins in tissues or at different subcellular localization in their native environment. However, capturing the spatial organization of proteins is challenging. Here, we present an innovative approach termed Spatial Proteomics through On-site Tissue-protein-labeling (SPOT), which combines the direct labeling of tissue proteins in situ on a slide and quantitative mass spectrometry for the profiling of spatially-resolved proteomics. MATERIALS AND METHODS: Efficacy of direct TMT labeling was investigated using seven types of sagittal mouse brain slides, including frozen tissues without staining, formalin-fixed paraffin-embedded (FFPE) tissues without staining, deparaffinized FFPE tissues, deparaffinized and decrosslinked FFPE tissues, and tissues with hematoxylin & eosin (H&E) staining, hematoxylin (H) staining, eosin (E) staining. The ability of SPOT to profile proteomes at a spatial resolution was further evaluated on a horizontal mouse brain slide with direct TMT labeling at eight different mouse brain regions. Finally, SPOT was applied to human prostate cancer tissues as well as a tissue microarray (TMA), where TMT tags were meticulously applied to confined regions based on the pathological annotations. After on-site direct tissue-protein-labeling, tissues were scraped off the slides and subject to standard TMT-based quantitative proteomics analysis. RESULTS: Tissue proteins on different types of mouse brain slides could be directly labeled with TMT tags. Moreover, the versatility of our direct-labeling approach extended to discerning specific mouse brain regions based on quantitative outcomes. The SPOT was further applied on both frozen tissues on slides and FFPE tissues on TMAs from prostate cancer tissues, where a distinct proteomic profile was observed among the regions with different Gleason scores. CONCLUSIONS: SPOT is a robust and versatile technique that allows comprehensive profiling of spatially-resolved proteomics across diverse types of tissue slides to advance our understanding of intricate molecular landscapes.