The glycoproteome remains severely understudied because of significant analytical challenges associated with glycoproteomics, the system-wide analysis of intact glycopeptides. This review introduces important structural aspects of protein N-glycosylation and summarizes the latest technological developments and applications in LC-MS/MS-based qualitative and quantitative N-glycoproteomics. These maturing technologies provide unique structural insights into the N-glycoproteome and its synthesis and regulation by complementing existing methods in glycoscience. Modern glycoproteomics is now sufficiently mature to initiate efforts to capture the molecular complexity displayed by the N-glycoproteome, opening exciting opportunities to increase our understanding of the functional roles of protein N-glycosylation in human health and disease.

Growing evidence indicates that the individualized and highly reproducible N-glycan repertoires on each protein glycosylation site modulate function. Relationships between protein structures and the resulting N-glycoforms have previously been observed, but remain to be quantitatively confirmed and examined in detail to define the responsible mechanisms in the conserved mammalian glycosylation machinery. Here, we investigate this relationship by manually extracting and analyzing quantitative and qualitative site-specific glycoprofiling data from 117 research papers. Specifically, N-glycan structural motifs were correlated with the structure of the protein carriers, focusing on the solvent accessibility of the individual glycosylation sites and the physicochemical properties of the surrounding polypeptide chains. In total, 474 glycosylation sites from 169 mammalian N-glycoproteins originating from different tissues/body fluids were investigated. It was confirmed statistically that the N-glycan type, degree of core fucosylation and branching are strongly influenced by the glycosylation site accessibility. For these three N-glycan features, glycosylation sites carrying highly processed glycans were significantly more solvent-accessible than those carrying less processed counterparts. The glycosylation site accessibilities could be linked to molecular signatures at the primary and secondary protein levels, most notably to the glycoprotein size and the proportion of glycosylation sites located in accessible β-turns. In addition, the subcellular location of the glycoproteins influenced the formation of the N-glycan structures. These data confirm that protein structures dictate site-specific formation of several features of N-glycan structures by affecting the biosynthetic pathway. Mammals have, as such, evolved mechanisms enabling proteins to influence the N-glycans they present to the extracellular environment.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometry based glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved ‘high-coverage’ and ‘high-accuracy’ glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.