A Micro-Quantitative and FFPE-Compatible Workflow for Immunohistochemistry-Guided Spatial Proteomic Analysis of Cellular Subpopulations Within the Tumor Microenvironment

作者：Junya Peng, Lu Ping, Ruikang Dun, Lu Liu, Y Shi, Ruizhe He, Qing Zhong, Yang Chen, Wenmin Tian, Yupei Zhao · 发表于：Bioengineering · 年份：2026 · DOI：10.3390/bioengineering13060678 · 研究领域：Advanced Proteomics Techniques and Applications、Single-cell and spatial transcriptomics、Advanced Biosensing Techniques and Applications

Understanding the spatial proteomic landscape of human tumors is essential for dissecting cellular heterogeneity and microenvironmental interactions in cancer biology. Traditional bulk proteomic approaches, however, obscure spatial information and average out signals from distinct cell populations. Here, we present a detailed and reproducible micro-quantitative protocol for spatially resolved proteomic analysis of specific cellular subpopulations isolated from immunohistochemistry (IHC)-labeled formalin-fixed paraffin-embedded (FFPE) tissue sections using laser microdissection (LMD). By combining IHC staining to visually define phenotypically distinct cells within preserved tissue architecture and precise LMD capture, approximately 6000 target cells can be isolated per sample for downstream proteomic quantification. Despite the ultra-low input, optimized lysis and digestion steps ensure consistent peptide recovery and highly reproducible label-free LC-MS/MS data across replicates. Integrating immunohistochemistry staining-guided spatial sampling with ultrasensitive quantitative proteomics, this workflow enables reliable cell-type-specific profiling directly within human tumor tissues. The protocol bridges histopathology and proteomics, offering a practical framework for translational research exploring spatial protein signatures and tumor microenvironmental heterogeneity.