Optimization of MALDI-TOF mass spectrometry imaging for the visualization and comparison of peptide distributions in dry-cured ham muscle fibers

•Comprehensive MALDI MSI sample preparation ensued optimized experimental settings.•Peptide distribution was visualized on Istrian dry-cured ham slices.•Differentially resolved spatial data reveals slight changes in proteolysis outcome.•MALDI MSI and PCA proved useful in a batch quality control.•Potential Istrian dry-cured ham peptide biomarkers were identified. In this article, a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI)-based study was designed in order to selectively map and compare the peptides present in slices of Biceps femoris, Istrian dry-cured ham muscles, from the same production batch. A systematic sample preparation process was optimized, which comprises embedding samples of Biceps femoris, cryo-sectioning, glass slide mounting, a nine-step washing protocol, MALDI matrix sublimation and recrystallization. This process efficiently preserved sample morphology and removed the high salt and lipid content, which was present in the samples as a result of the dry-curing production process. We show that MALDI MSI, in combination with principal component analysis, can be used to monitor subtle changes in proteolysis outcome within the same dry-cured ham muscle type, resulting from differentially resolved spatial data. The peptides identified in Istrian dry-cured ham may therefore be studied further, as putative biomarkers for this specific product.