The Campylobacter jejuni pgl locus encodes an N‐linked protein glycosylation machinery that can be functionally transferred into Escherichia coli. In this system, we analyzed the elements in the C. jejuni N‐glycoprotein AcrA required for accepting an N‐glycan. We found that the eukaryotic primary consensus sequence for N‐glycosylation is N terminally extended to D/E‐Y‐N‐X‐S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N‐glycosylated when they were either artificially introduced or when they were present in non‐C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N‐glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in C. jejuni N‐glycoproteins. N‐glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the −2 position. Thus, bacterial N‐glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.

N-linked protein glycosylation is found in all domains of life. In eukaryotes, it is the most abundant protein modification of secretory and membrane proteins, and the process is coupled to protein translocation and folding. We found that in bacteria, N-glycosylation can occur independently of the protein translocation machinery. In an in vitro assay, bacterial oligosaccharyltransferase glycosylated a folded endogenous substrate protein with high efficiency and folded bovine ribonuclease A with low efficiency. Unfolding the eukaryotic substrate greatly increased glycosylation. We propose that in the bacterial system, glycosylation sites are located in flexible parts of folded proteins, whereas the eukaryotic cotranslational glycosylation evolved to a mechanism presenting the substrate in a flexible form before folding.

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.

Bacterial cultures can express proteins in high yields but cannot create mammalian N-glycoforms. Engineering of the glycosylation machinery of C. jejuni and its transfer into E. coli, combined with trimming and rebuilding of the N-linked glycan, now provides a robust route to glycoproteins. We describe a new method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the Campylobacter jejuni glycosylation machinery in Escherichia coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins.

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.