Spatial Metabolomics Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) Kidney Tissue: Workflow Development and Optimization

Background: Formalin fixation and paraffin embedding (FFPE) is the gold standard tissue preservation method in routine pathological diagnostics. However, formalin fixation induces formation of intra- and intermolecular crosslinks. These chemical modifications pose significant challenges for downstream spatial metabolomics imaging, requiring further investigation into the feasibility of molecular recovery from FFPE kidney tissue. Methods: The workflow for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of FFPE blocks is shown in Figure 1A. Sections from FFPE-preserved mouse kidney cortex were mounted on indium tin oxide (ITO)-coated slide in a cryostat. Autofluorescence image was taken prior to matrix application using 1,5-diaminonaphthalene (DAN). Slide was analyzed by MALDI coupled Q Exactive MS. Raw data were converted and annotated spatially via METASPACE, incorporating formalin-related adducts (+CH2O, +CH2, +CH2OH) to account for fixation-induced modifications. Results: With established workflow, homogenous matrix coverage and robust signal intensity were achieved. We successfully identified D-4'-phosphopantothenate with elevated signal intensity localized to the glomeruli of kidney (Fig. 1B), as previously found in frozen sections of mouse and human kidneys, highlighting the capability of this workflow to retain and reveal spatial metabolite distribution. METASPACE annotation confirmed the presence of amino acid crosslinking products by formalin fixation, with approximately 60 dipeptides identified (representative image Fig. 1C), and strong on-tissue signals for disease-relevant small molecules such as adenine (Fig. 1D). Metabolite identities were validated through MS/MS fragmentation. Conclusion: Our study demonstrates a reliable MALDI-MSI workflow for spatial metabolomic profiling of FFPE kidney tissue, enabling precise localization of discrete metabolites within distinct regions of preserved tissue architecture. Results emphasize the potential of FFPE-preserved tissues for selected metabolite profiling, significantly expanding the utility of archived clinical specimens and enhancing the scope of MALDI-MSI for translational research. Funding: NIDDK Support