A general approach for glycoprotein analysis: glycopeptide analysis by LC/MS with HILIC enrichment and glycan permethylation profiling

N‐linked glycosylation is one of the most common protein modifications observed in eukaryotic systems. This modification is important in many biological functions and it attracts increasing interest in basic research and the pharmaceutical industry. Glycosylation often presents as a stochastic distribution of related structural entities. Not only must the range of glycan structures be characterized but their position on the polypeptide backbone must also be determined. We have developed a mass spectrometry based glycomics platform. This platform provides positional information allowing mapping of specific glycoforms to N‐glycosylation sequons as well as a semi‐quantitative analysis of total protein glycosylation. Using publically available software tools these data are processed and the glycans mapped to the three dimensional surface of glycoproteins using the NCBI structural database and protein alignment tools. This platform should allow for a better understanding of protein N‐glycosylation and for demonstration of glycosylation differences through semi‐quantitative comparison at the free glycan level. It should allow for a greater understanding of N‐glycosylation processes in the basic science setting and allow for greater control of the manufacturing process and a better understanding of how glycosylation affects the performance of glycoprotein biologics in the pharmaceutical setting.