Lagou, M. K. | bioRxiv Science

bioRxiv2025

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

The ability to identify spatially resolved proteomes has advanced markedly in recent years, yet integrating definitive protein identification with precise spatial localization in a single workflow remains a challenge. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables antibody-free mapping of proteins directly in tissue sections, but its capacity for unambiguous protein 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 that are critical to thymic function. Using this pipeline, we reveal spatiotemporal changes in proteins involved in cell migration, cytoskeletal remodeling, and endogenous thymic regeneration. Notably, we identify distinct spatial shifts in Nucleoprotein TPR and Tubulin-associated chaperone A (TBCA), corresponding to chemotherapy-driven architectural remodeling. From a translational perspective, these findings highlight pathways and candidate 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 and other tissues, broadening the potential of translational proteomic research.

cancer biology↗

bioRxiv2024

Morphometric Analysis of the Thymic Epithelial Cell (TEC) Network Using Integrated and Orthogonal Digital Pathology Approaches

The thymus, a central primary lymphoid organ of the immune system, plays a key role in T cell development. Surprisingly, the thymus is quite neglected with regards to standardized pathology approaches and practices for assessing structure and function. Most studies use multispectral flow cytometry to define the dynamic composition of the thymus at the cell population level, but they are limited by lack of contextual insight. This knowledge gap hinders our understanding of various thymic conditions and pathologies, particularly how they affect thymic architecture, and subsequently, immune competence. Here, we introduce a digital pathology pipeline to address these challenges. Our approach can be coupled to analytical algorithms and utilizes rationalized morphometric assessments of thymic tissue, ranging from tissue-wide down to microanatomical and ultrastructural levels. This pipeline enables the quantitative assessment of putative changes and adaptations of thymic structure to stimuli, offering valuable insights into the pathophysiology of thymic disorders. This versatile pipeline can be applied to a wide range of conditions that may directly or indirectly affect thymic structure, ranging from various cytotoxic stimuli inducing acute thymic involution to autoimmune diseases, such as myasthenia gravis. Here, we demonstrate applicability of the method in a mouse model of age-dependent thymic involution, both by confirming established knowledge, and by providing novel insights on intrathymic remodeling in the aged thymus. Our orthogonal pipeline, with its high versatility and depth of analysis, promises to be a valuable and practical toolset for both basic and translational immunology laboratories investigating thymic function and disease.

immunology↗