Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) has been used to generate spatial maps of lipids, metabolites, peptides, proteins, and glycans in tissues
however, its use for mapping extracellular matrix (ECM) protein distributions is underexplored. ECM proteins play a major role in various pathological conditions, and changes in their spatial distributions affect the function and morphology of cells within tissues. ECM protein detection is challenging because they are large, insoluble, and undergo various post-translational modifications, such as glycosylation. We describe here decellularization of tissue sections coupled with serial enzymatic digestions with PNGaseF and trypsin to improve ECM protein detection in MALDI-MSI without disrupting ECM architecture. Decellularization leads to a 3-fold increase in the number of proteins that are measured by MALDI-MSI. We also introduce a binary colocalization method to improve protein identification, which increases the number of proteins that are confidently detected. Together, these methods enhance the spatial mapping of ECM proteins by MALDI-MSI.