Navigating the maze of mass spectra: a machine-learning guide to identifying diagnostic ions in O-glycan analysis

Structural details of oligosaccharides, or glycans, often carry biological relevance, which is why they are typically elucidated using tandem mass spectrometry. Common approaches to distinguish isomers rely on diagnostic glycan fragments for annotating topologies or linkages. Diagnostic fragments are often only known informally among practitioners or stem from individual studies, with unclear validity or generalizability, causing annotation heterogeneity and hampering new analysts. Drawing on a curated set of 237,000 O-glycomics spectra, we here present a rule-based machine learning workflow to uncover quantifiably valid and generalizable diagnostic fragments. This results in fragmentation rules to robustly distinguish common O-glycan isomers for reduced glycans in negative ion mode. We envision this resource to improve glycan annotation accuracy and concomitantly make annotations more transparent and homogeneous across analysts.