Fig. 2

Operational procedure of spatial proteomics mass spectrometry methods. 1. Sample Collection: Different tissue samples are collected. 2. Slice Staining: Tissue samples were sectioned and mounted onto glass slides. Following natural air-drying, appropriate staining techniques, such as Hematoxylin and Eosin (H&E) staining, immunohistochemical staining, Masson’s trichrome staining, etc., were chosen based on experimental design. 3. Laser Fiber cutting: The laser microdissection system is used for precise localization and microdissectionof the regions of interest under a microscope. 4. Protein Extraction: were extracted using protease or protein extraction buffer.. 5. Protein Digestion: The extracted proteins were subjected to digestion to release peptide fragments. 6. LC–MS/MS: The peptide solution is injected into the liquid chromatography system, where separation of different peptide segments is achieved using a chromatographic column. Subsequently, the separated peptide segments are introduced into the mass spectrometer, where they are ionized. The mass spectrometer evaluates the protein mass and relative abundance by analyzing the mass-to-charge ratio of ions in the peptide segments. 7. Raw Data: The data can be preprocessed through baseline correction, peak fitting, and other methods. 8. Statistical Analysis: The utilization of data visualization tools for differential analysis on raw data allows for the revelation of information concerning the distribution and abundance of specific proteins across different spatial context