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<p>Filter-aided expansion proteomics (FAXP) is a spatial proteomics approach designed for high-resolution analysis of formalin-fixed, paraffin-embedded (FFPE) tissues. Here we describe the integration of hydrogel-based tissue expansion with mass spectrometry, enabling isotropic expansion and robust protein retention while preserving spatial features. The FAXP workflow consists of several sequential steps, including tissue section dewaxing, in situ protein anchoring, hydrogel embedding, homogenization, staining, isotropic expansion, microdissection and filter-aided in-gel digestion to maximize peptide recovery. The Protocol integrates laser capture microdissection, enabling the precise isolation of single cells and subcellular components for subcellular spatial proteomics analysis. The approach achieves up to a fivefold linear expansion factor of FFPE tissue, including extracellular matrix-rich samples such as colorectal cancer, with less than 6% distortion, enabling the identification of an average of 2,368 proteins from single mouse liver nucleus shape and 3,312 proteins from single mouse liver cell shape using an Astral mass spectrometer. The method is compatible with diverse tissue types, including extracellular matrix-rich specimens, and integrates seamlessly with imaging workflows, such as immunostaining, for spatially resolved proteomic analysis. FAXP enables researchers to obtain comprehensive proteomic profiles with strong reproducibility and high sensitivity. The entire workflow takes ~27 h and requires only commercially available reagents and supplies and is thus accessible for researchers with intermediate expertise in tissue processing, microscopy and proteomics. FAXP can advance spatial proteomics-based studies, in particular of cancer heterogeneity, neurodegenerative diseases and cellular microenvironments within FFPE tissues, including archival clinical samples.</p>

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Filter-aided expansion proteomics for the spatial analysis of single cells and organelles in FFPE tissue samples

  • 期刊论文

  • 发表日期：

    2025年10月9日

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  • Zhen Dong,
  • Chunlong Wu,
  • Jiayi Chen,
  • Wenhao Jiang,
  • Kiryl D. Piatkevich,
  • Yi Zhu,
  • Tiannan Guo

摘要

Filter-aided expansion proteomics (FAXP) is a spatial proteomics approach designed for high-resolution analysis of formalin-fixed, paraffin-embedded (FFPE) tissues. Here we describe the integration of hydrogel-based tissue expansion with mass spectrometry, enabling isotropic expansion and robust protein retention while preserving spatial features. The FAXP workflow consists of several sequential steps, including tissue section dewaxing, in situ protein anchoring, hydrogel embedding, homogenization, staining, isotropic expansion, microdissection and filter-aided in-gel digestion to maximize peptide recovery. The Protocol integrates laser capture microdissection, enabling the precise isolation of single cells and subcellular components for subcellular spatial proteomics analysis. The approach achieves up to a fivefold linear expansion factor of FFPE tissue, including extracellular matrix-rich samples such as colorectal cancer, with less than 6% distortion, enabling the identification of an average of 2,368 proteins from single mouse liver nucleus shape and 3,312 proteins from single mouse liver cell shape using an Astral mass spectrometer. The method is compatible with diverse tissue types, including extracellular matrix-rich specimens, and integrates seamlessly with imaging workflows, such as immunostaining, for spatially resolved proteomic analysis. FAXP enables researchers to obtain comprehensive proteomic profiles with strong reproducibility and high sensitivity. The entire workflow takes ~27 h and requires only commercially available reagents and supplies and is thus accessible for researchers with intermediate expertise in tissue processing, microscopy and proteomics. FAXP can advance spatial proteomics-based studies, in particular of cancer heterogeneity, neurodegenerative diseases and cellular microenvironments within FFPE tissues, including archival clinical samples.