Identifying space-resolved proteins of the murine thymus, by combining MALDI-MSI and proteomics

Identifying spatially resolved proteomes has advanced markedly, yet integrating definitive protein identification with precise spatial localization in a single workflow remains challenging. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables antibody-free mapping of proteins in tissue sections, but its capacity for unambiguous identification is limited. Here, we present a combined MALDI-MSI and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach to map protein localization and track spatial changes in murine thymus during chemotherapy-induced involution and regeneration. Our workflow incorporates a scoring algorithm (pepBridge) that aligns MALDI-MSI molecular signals with LC-MS/MS identifications, enabling confident assignment of proteins, critical to thymic function. Using this pipeline, we reveal spatiotemporal changes in proteins involved in cell migration, cytoskeletal remodeling, and thymic regeneration. Notably, we identify distinct spatial shifts in nucleoprotein TPR and tubulin-associated chaperone A (TBCA), corresponding to chemotherapy-driven architectural remodeling. Translationally, these findings highlight pathways and targets to promote immune recovery in pediatric cancer patients undergoing cytoreductive therapy. Analytically, this framework advances spatial proteomics by enabling high-confidence protein identification in lymphoid tissues, broadening the potential of translational proteomic research.