Evaluating the Effects of Acquisition and Search Parameters on Repository-Scale MS/MS Matching

Abstract

Tandem mass spectrometry (MS/MS) spectral matching is increasingly performed at the repository scale, where tools such as MASST enable a query spectrum to be compared against heterogeneous public data. Unlike analogous genomic-based searches, repository-scale MS/MS matching lacks integrated false discovery rate (FDR) reporting, leaving parameter selection entirely to user discretion. Here, we evaluate how acquisition factors and analysis filters jointly affect MS/MS matching using a curated library of 40 metabolites spanning ten chemical classes. In this study, we find that normalized collision energies from 30 to 50% balance sufficient fragmentation against loss of larger diagnostic ions, producing the most structurally informative spectra and yielding the highest proportion of repository matches. Using a fragmentation-based decoy library, we show that six minimum matching peaks achieve <1% FDR across the entire cosine threshold range tested (0.7–1.0), while fewer matching peaks require increasingly stringent cosine thresholds. These findings provide practical, benchmarking guidance for user-provided acquisition and search parameters in MASST searches.