From identification to quantification: A step-by-step workflow for intact glycopeptide analysis by mass spectrometry

Protein glycosylation is a universal post-translational modification that plays vital roles in regulating protein function, stability, and cell-cell communication. Consequently, aberrant glycosylation is implicated in various diseases. Characterizing glycoproteomic changes holds significant potential for elucidating their underlying mechanisms and advancing clinical applications. Mass spectrometry (MS) enables in-depth profiling of intact glycopeptides, providing information on both site-specific glycan compositions alongside corresponding peptide backbones. In this tutorial, we present an optimized workflow for the qualitative and quantitative analysis of intact glycopeptides using MS, building upon our recent work. We detail practical protocols for sample preparation and intact glycopeptide enrichment, compare label-free and label-based quantification methods, describe fragmentation strategies with optimized parameters, and review available tools for data analysis. Our goal is to support reproducible, high-quality MS-based glycoproteomic studies across diverse biological and clinical applications.