Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycansand their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycanand peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteomecharacterization, especially for N- glycopeptides, which can have much more branched glycanside chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycanand peptide fragments in one single spectrum, enabling complete informationto be gathered and great microheterogeneity details to be revealed. Fetuinwas first utilized to prove the applicability with 19 glycopeptidesand corresponding five glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N- glycoproteomicsin rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N- glycopeptidesand N- glycoproteomics. We also anticipate this approach can be readily applied to large-scale O- glycoproteomecharacterization.