Navigating the Complex Landscape of N-Glycoproteomics: Guidelines for Large-Scale Data Processing

ABSTRACT: Large-scale N-glycoproteomics remains challenging because the structural complexity and heterogeneity of glycans can lead to frequent misassignments in automated MS2-based glycopeptide annotation. In particular, isobaric and isomeric monosaccharide combinations, ammonia adducts, carbamidomethylation-related artifacts, and oxidative mass variants complicate confident glycopeptide identification, and errors in monoisotopic peak assignment can further increase misannotation, often necessitating careful evaluation beyond MS2 scoring alone. Here, human serum N-glycopeptides were analyzed using an integrated data-processing strategy designed to improve the accuracy and consistency of large-scale glycoproteomics data analysis. Monocle was used to improve monoisotopic precursor assignment prior to database searching, followed by glycopeptide identification with Byonic. Candidate glycopeptides were then critically evaluated using a Skyline-based curation workflow that integrated orthogonal evidence from accurate mass, isotopic overlap, sialic acid-dependent retention-time clustering, and glycosite context, together with supporting fragment-ion information where relevant. Applied to a human serum case study comprising 52 samples, this workflow enabled systematic curation of a large candidate set generated from LC-MS/MS data. In total, 3,071 candidate N-glycopeptides were assessed in Skyline after MS2-based searching and filtering. Validation refuted 1,722 MS2-based candidate IDs that were inconsistent with chromatographic and/or precursor m/z-level evidence. The final curated dataset comprised 1,436 N-glycopeptides across 148 N-glycosylation sites on 58 glycoproteins. Together, these results demonstrate how orthogonal MS1/RT-guided curation can substantially improve confidence, accuracy, and completeness of large-scale site-resolved human serum N-glycoproteomics.

ORGANISM(S): Homo Sapiens

SUBMITTER: Juan C. Rojas Echeverri  

PROVIDER: PXD077279 | panorama | Wed Oct 14 00:00:00 BST 2026

REPOSITORIES: PanoramaPublic