Spatial proteomics for the analysis of host-pathogen interactions in mice lungs infected with <i>Aspergillus fumigatus</i>

Abstract Understanding host-pathogen interactions at the molecular level requires methods capable of linking spatial context with proteomic information. Here, we present an integrated workflow combining matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) and laser microdissection (LMD)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate Aspergillus fumigatus infection in murine lung tissue. Consecutive formalin-fixed, paraffin-embedded (FFPE) tissue sections were used for spatially resolved MALDI-MSI and subsequent LC-MS/MS analysis of laser-microdissected fungal-infected and non-infected regions. MALDI-MSI revealed reproducible m/z features specifically associated with infected areas. Corresponding tissue microregions were microdissected and analysed by proteomics to identify candidate proteins underlying these spatial signals. Comparative proteomics of fungal-infected with non-infected alveolar lung regions via LC-MS/MS identified host proteins involved in leukocyte recruitment, inflammatory signaling, and reactive oxygen species formation, including a 424-fold increase in formyl peptide receptor 2 (Fpr2) during fungal invasion of the lungs. Fungal regions were also enriched in proteins encoded by the gliotoxin biosynthetic gene cluster. Spatial and proteomic data were linked by matching theoretical peptide-adduct masses to MALDI-MSI features, using a semi-quantitative scoring system to prioritize protein assignments. Fungal regions showed contributions from both host and pathogen proteins. This workflow establishes a conceptual basis for spatial proteomics of host-pathogen-interactions in fungal infections and enables association of characteristic m/z signals with plausible protein candidates.