Home » Abstracts » Imaging Proteomics » Linking spatial to functional proteomics on heterogeneous samples: The “pixel-by-pixel” shotgun proteomic approach

Linking spatial to functional proteomics on heterogeneous samples: The “pixel-by-pixel” shotgun proteomic approach

Imaging Proteomics Interactomics & Spatial Omics

Abstract #296 Posted on28 March 202512 May 2025 12 May 2025 10h42

Abstract

Proteomics of biological tissues faces challenges due to sample heterogeneity. Mass spectrometry imaging offers spatial information with limited functional insights, while laser capture microdissection shotgun proteomics provides detailed proteome data but sacrifices spatial resolution. We propose a technique, called pixel-by-pixel shotgun proteomics (Pixel2 Proteomics, P2P), combining laser microdissection with proteomics.

This approach was tested on patient-derived xenografts from triple-negative breast cancer tissues, both treated and untreated with anti-cancer drugs. Tissues were initially preserved in formalin-fixed paraffin-embedded (FFPE) form and sectioned into 5 µm and 10 µm slices.

The 10 µm slices underwent matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) following on-tissue digestion with trypsin or PNGase to release peptides and glycans. Hierarchical clustering of MSI pixels revealed heterogeneous regions. The 5 µm tissues were microdissected on polyethylene naphthalate (PEN) slides into areas of 2,500 µm² (10-20 cells), following a systematic grid pattern. Subsequent microdissection of both drug-treated and untreated tissues led to the identification of 1,000 to 3,000 proteins per pixel. This analysis highlighted proteins specific to peripheral, inflammatory, and hypoxic regions and the impact of the anti-tumor treatment.

This method provides detailed spatial and functional insights into tissue heterogeneity.

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This approach was tested on patient-derived xenografts from triple-negative breast cancer tissues, both treated and untreated with anti-cancer drugs. Tissues were initially preserved in formalin-fixed paraffin-embedded (FFPE) form and sectioned into 5 µm and 10 µm slices.

The 10 µm slices underwent matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) following on-tissue digestion with trypsin or PNGase to release peptides and glycans. Hierarchical clustering of MSI pixels revealed heterogeneous regions. The 5 µm tissues were microdissected on polyethylene naphthalate (PEN) slides into areas of 2,500 µm² (10-20 cells), following a systematic grid pattern. Subsequent microdissection of both drug-treated and untreated tissues led to the identification of 1,000 to 3,000 proteins per pixel. This analysis highlighted proteins specific to peripheral, inflammatory, and hypoxic regions and the impact of the anti-tumor treatment.

This method provides detailed spatial and functional insights into tissue heterogeneity.

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