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Abstract

Glycosylation is one of the most common and complex post-translation modifications that influence the structural and functional properties of proteins. Glycoproteins are highly heterogeneous and exhibit site- and protein-specific expression differences. Mass spectrometry in combination with liquid chromatography has emerged as the most powerful tool for the comprehensive characterization of glycosylation. The analysis of intact glycopeptides has emerged as a promising strategy to analyze glycoproteins for their glycan heterogeneity at both protein- and site-specific levels. Nevertheless, intact glycopeptide characterization is challenging as elucidation of the glycan and peptide moieties requires specific sample preparation workflows that, combined with the tandem mass spectrometry approach, enable the identification of single glycopeptide species. In this chapter, we provide a detailed description of the methods that include procedures for (i) proteolytic digestion using specific proteases, (ii) optional glycopeptide enrichment using hydrophilic interaction liquid chromatography, (iii) nano-LC-MS/MS analysis of glycopeptides, and (iv) data analysis for identification of glycopeptides. Together, our workflow provides a framework for the system-wide site-specific analysis of N- and O-glycopeptides derived from complex biological or clinical samples.