Full Scan enhanced Dynamic Range MS improves metabolite coverage and cancer cell-line discrimination in untargeted metabolomics

Metabolite detection with mass spectrometry (MS) in untargeted metabolomics is limited by the wide concentration range of metabolites, where high-abundance signals dominate MS1 scans and suppress detection of low-abundance features. This reduces metabolite coverage and obscures biologically relevant signals, particularly in complex cellular systems. Full Scan enhanced Dynamic Range (eDR) MS addresses these limitations by partitioning the MS1 mass range into multiple subscans and mass windows, reducing saturation effects from dominant ions. Here, we systematically evaluate different eDR acquisition strategies for untargeted metabolomics. Across four hepatocellular carcinoma cell lines, Full Scan eDR MS increased detectable features up to [~]3.5-fold compared to Full Scan MS. Among equidistant window configurations, 12 windows yielded the highest feature count and broadest dynamic range, while custom window distributions further improved detection in ion-dense regions. In particular, allocating smaller window sizes to the low m/z region selectively increased detection of low-mass features while preserving performance for higher mass ions. Full Scan eDR MS also improved data quality, reducing variation and increasing signal-to-noise ratios, especially for low-abundance metabolites. MS2 coverage and metabolite identifications increased substantially, resulting in unique detection of cancer-relevant metabolites. Importantly, the increased depth of metabolite detection enabled improved discrimination between cancer cell lines, supporting deeper interrogation of metabolic heterogeneity. Overall, these results establish Full Scan eDR MS as a flexible strategy to improve sensitivity and metabolome coverage in untargeted metabolomics. Customization of window size and distribution enable targeted expansion of dynamic range within predefined mass regions, allowing MS acquisition to be tailored to sample complexity and metabolites of interest.