Reproducibility Limits of Public MS/MS Spectral Libraries: Irreproducibility, Outlier Spectra, and Indistinguishable Compounds Across One Million Reference Spectra
Year: 2026
Type: preprint
Abstract: Compound annotation in untargeted metabolomics relies on matching an experimental MS/MS spectrum against a reference library, and the quality of such libraries is routinely summarized by the proportion of spectrum pairs exceeding a similarity threshold. We show that this class of metric is structurally invalid. Analyzing 1,022,449 spectra covering 66,843 compounds from GNPS, MS-DIAL, and MassBank of North America, we compute all within- and between-compound pairwise similarities and demonstrate that proportion-based consistency estimates are confounded by sampling depth as a matter of construction: the apparent high-consistency rate falls from 91% for compound groups supported by a single comparison to 5% for groups supported by more than one hundred. Consequently, any comparison of libraries, instrument platforms, or acquisition conditions that does not control depth measures coverage rather than quality — an apparent advantage of one instrument platform over another narrows substantially once depth is matched. We further identify a resolution floor that no improvement in library curation can remove: 31.7% of compounds share near-identical spectra (cosine ≥ 0.9) with at least one structurally distinct compound, so for these the identification is underdetermined by spectral matching alone. Against this corrected framework, 49.6% of 12,613,259 same-compound comparisons fall below the conventional 0.7 threshold and 6.4% of spectra behave as outliers within their own compound group. We release the collection annotated with spectrum-, group-, and compound-level metrics, each reported alongside the evidence supporting it rather than as a bare proportion, together with the pipeline as an open-source R package.
Source: ChemRxiv
Author: Yongseok Kim
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7204993907