Abstract

Glycopeptide enrichment remains a cornerstone in glycoproteomics, but bias and reproducibility issues continue to hinder biological insight and clinical translation. Using curated glycoproteomics datasets and machine learning, we trained a glycopeptide classifier to promptly recognize N-glycopeptide precursor ions in peptide mixtures through mass defect signatures. Integration of the classifier into a data-dependent acquisition framework facilitated efficient and unbiased real-time prediction of N-glycopeptides directly from serum opening avenues for enrichment-free glycoproteomics.