Authentic Spatial Metabolomics by Avoiding Hidden Pitfalls in MSI Workflows.

Mass spectrometry imaging (MSI) has emerged as a powerful analytical tool for visualizing the spatial distribution of endogenous molecules in complex biological tissues. However, achieving authentic and reproducible metabolic maps remains challenging due to pitfalls in sample preparation and instrumental limitations that are often overlooked. Herein, based on practical evaluation, we delineate four critical but frequently overlooked pitfalls in MSI workflows: metabolic quenching, matrix effects, mass resolving power, and quadrupole isolation width. We demonstrate that thaw-mounting during traditional tissue preparation reactivates metabolic enzymes, which can be mitigated by rapid heat stabilization above 100 °C. Region-specific matrix effects causing uneven ionization could be corrected by normalization against isotope-labeled internal standards. Using incremental resolving power, we reveal that a single nominal m/z can represent multiple isobaric species with distinct spatial patterns, underscoring the necessity of ultrahigh resolution. Furthermore, we introduce a spatial correlation analysis that deconvolutes chimeric MS/MS spectra from coisolated precursors, enabling correct assignment of isobaric metabolites (e.g., choline and aminobutyric acid). Collectively, this study identifies four pitfalls and provides actionable strategies to overcome them, offering a practical framework for more reliable spatial metabolomics in preclinical and clinical research.