Going a Layer Deeper: Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) to Analyze...

As soft tissues degrade, structural features used to interpret post-mortem change are progressively lost, limiting the information available from morphology alone. Molecular approaches capture biochemical transformations during decomposition, yet most rely on homogenized tissues that obscure spatial variation across distinct tissue layers. Here, we present a layer-resolved mass spectrometry framework combining matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiling across multiple donors with a single case study using MALDI MS-imaging (MSI). This approach was used to examine how decomposition progresses within distinct soft tissue layers (skin, adipose, and muscle) in human donors. Hematoxylin and eosin (H&E) staining was used in conjunction with MSI to assess the preservation of tissue structure over time and to verify the identity of anatomical layers. MALDI-MS profiling revealed clear molecular differences among skin, adipose, and muscle, with each layer maintaining a distinct lipid profile and showing unique sets of accumulated degree days (ADD)-correlated features despite progressive structural loss. MALDI-MSI, performed on a representative donor, further showed how decomposition-associated molecules redistributed across tissue layers over time, highlighting spatial patterns not evident from morphology alone. This study demonstrates that layer-resolved MALDI-based profiling, complemented by exploratory imaging can reveal persistent molecular signatures during soft-tissue decomposition, providing a refined framework for identifying tissue-specific biomarkers with potential value for future post-mortem interval (PMI) estimation.