![]() The end result is a multidimensional, information-rich dataset containing metabolic features from normal and diseased tissues 24, 30, 31, 32. It is capable of high sampling aptitude with the ability to record tens of thousands of pixel-mapped spectra from a single tissue section coupled with spatial resolution 26, 27, 28, 29. MALDI-MSI utilizes robotic precision micromovement coupled to a Nd:YAG laser for spatial profiling of both organic and biological matter 23, 24, 25. To address this limitation, a new technique was recently developed employing enzyme-assisted matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) that can simultaneously perform spatial profiling of both glycogen and N-linked glycans from formalin-fixed paraffin-embedded (FFPE) mammalian tissue sections in situ, preserving important anatomical regions 15, 22. Oligomerization of simple sugars to glycogen results in increased polarity and decreased solubility therefore, quantitative, and spatial analyses of complex carbohydrates has been challenging. They are highly concentrated in pulmonary fibrosis and are part of the extracellular collagen, proteoglycan, and N-linked glycan matrix to support fibroblast growth and contribute to remodeled lung architecture, function, and hindering gas exchange 20, 21. N-linked glycans are especially critical as structural components for anatomical regions, and are vital for immune modulation and organ function 19. Together, complex carbohydrates modulate a myriad of cellular functions including bioenergetics, epigenetics, protein-ligand binding, membrane transport activity, and protein turnover 18. Anabolic pathways for the biosynthesis of glycogen and N-linked glycans span multiple cellular compartments including the nucleus, cytoplasm, endoplasmic reticulum, Golgi, and the plasma membrane 14, 15, 16, 17. To this end, in situ-based spatial technologies are beginning to emerge that can be mapped to anatomical regions, bridging the gap between genotype to phenotype analysis 12, 13.Ĭomplex carbohydrates such as N-linked glycans and glycogen are dynamic macrometabolites that impact complex and intertwined biochemical pathways 14, 15. Current single-cell approaches include cell-separation-based methods that are focused on molecular features such as proteins, lipids, and metabolites 9, 10, 11. The scRNAseq workflow has allowed researchers to study complex cellular heterogeneity from diseased and healthy tissues, revealing complex and rare cell populations, while uncovering regulatory relationships and tracking distinct cell lineages during development 5, 6, 7, 8. The resolution of single-cell RNA sequencing (scRNAseq) technology has impressive resolution that allows interrogation of tissue complexity and interactive networks at a cellular level 1, 2, 3, 4. Recent improvements in RNA sequencing technologies that allow the detailed analysis of a single isolated cell from host tissues is a major advancement in achieving this goal. Mapping genotype to phenotype with near single-cell resolution and spatial definition is a major innovation in next-generation life science techniques. In summary, our study provides a roadmap to leverage spatial metabolomics to understand foundational biology in pulmonary diseases. Collectively, we provide conclusive evidence that lysosomal utilization of glycogen is required for pulmonary fibrosis progression. Both mouse models displayed blunted N-linked glycan levels and nearly 90% reduction in endpoint fibrosis when compared to WT animals. To test our hypothesis, we induced pulmonary fibrosis in two different mouse models with lysosomal glycogen utilization deficiency. ![]() Using metabolic features identified from this pipeline, we hypothesize that metabolic channeling between glycogen and N-linked glycans is a critical metabolic process favoring pulmonary fibrosis progression. ![]() Here, we demonstrate the application of high-dimensionality reduction/spatial clustering and histopathological annotation of matrix assisted laser desorption/ionization imaging datasets to assess tissue metabolic heterogeneity in human lung diseases. Matrix assisted laser desorption/ionization imaging has greatly improved our understanding of spatial biology, however a robust bioinformatic pipeline for data analysis is lacking. Nature Communications volume 14, Article number: 2759 ( 2023) Spatial metabolomics reveals glycogen as an actionable target for pulmonary fibrosis
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