Identifying Space-Resolved Proteins of the Murine Thymus, by Combining MALDI Mass Spectrometry Imaging and Proteomics

The identification of spatially resolved proteomes has recently seen significant breakthroughs, yet challenges persist, particularly in integrating protein identification with precise spatial localiza-tion within a single experimental workflow. Matrix-assisted laser desorption ionization mass spec-trometry imaging (MALDI-MSI) enables spatial protein mapping on tissue sections without the need for antibodies. While MALDI-MSI addresses several obstacles in proteomic mapping with spatial resolution, it remains limited in its capacity for definitive protein identification. To over-come these limitations, this study introduces a novel approach combining MALDI-MSI with liquid chromatography-mass spectrometry (LC-MS/MS) to map protein localization and spatial chang-es in thymic tissue. Using a mouse model of chemotherapy-induced thymic involution with time-dependent regeneration kinetics, we evaluated the capability of this pipeline to resolve proteomic changes in a spatiotemporal manner. Our approach incorporates a scoring system to align mo-lecular signals from MALDI-MSI with proteomic data, enabling precise identification of proteins critical for thymic function. The results reveal key changes in protein expression, particularly in regions associated with cell migration, cytoskeletal remodeling, and endogenous thymic regen-eration. By analyzing the spatial distribution of proteins such as Keratin 8 (KRT8), Nucleoporin TPR, Cysteine- and Glycine-Rich Protein 3 (CSPR3), and Tubulin-Associated Chaperone-A (TBCA), we observed distinct shifts in thymic compartment architecture, underscoring the im-pact of chemotherapy on thymic tissue structure. From a translational viewpoint, this approach identifies new pathways and targetable candidates to enhance immune cell recovery in pediatric cancer patients undergoing cytoreductive treatments. From an analytical perspective, it provides an innovative framework for visualizing protein distribution in lymphoid and other tissues, signifi-cantly advancing the potential for translational proteomic research.