Recently gained knowledge on glycosylation disorders highlights the involvement of protein glycosylation in health and disease. The glycosylation sites in the amino acid sequences of glycoproteins constitute the primary information needed for understanding the molecular pathology and structure-function relationships. The mass spectrometry of glycopeptides plays a central role in elucidating the structural issues of glycoproteins, and is currently effective in the N-glycosylation of large glycoproteins as well. In contrast, site-specific analysis of glycopeptides containing O-glycans remains a formidable task. Unlike N-glycosylation, there is no reliable amino acid consensus sequence that predicts O-glycosylation, because of the presence of multiple N-acetylgalactosaminyltransferase isozymes, overlapping of their substrate specificities, and the clustering of attachment sites in a small region of the target glycoprotein. Additionally, the high proline, as well as serine and threonine, contents in the vicinities of glycosylated sites prevent collision-induced dissociation/ tandem mass spectrometry from traversing the entire length of the peptide sequence. In this review, various approaches such as glycopeptide enrichment, deglycosylation, β-elimination/Michael addition, and fixed-charge derivatization, all of which support conventional low-energy collision are summarized along with electron capture (transfer) dissociation-a new mode of peptide fragmentation.
Analysis of the carbohydrate heterogeneity of glycoprotein-based substances is crucial for establishing the nomenclature and definition of biological substances, ensuring consistency in the quality of these products, comparatively assessing the products obtained after the implementation of changes in the manufacturing process, and developing biosimilar or follow-on biological products. Liquid chromatography/mass spectrometry is recognized as one of the most useful techniques for analyzing the carbohydrate heterogeneity of glycoprotein substances. Here, we demonstrate the utility of LC/MS for analyzing the carbohydrate heterogeneity by using some representative glycoproteins such as tissue-plasminogen activator, a monoclonal antibody, the follicle-stimulating hormone, and human chorionic gonadotropin. Further, we demonstrate that MS-based glycoprotein analysis has potential applications in glycomics.
Growing evidence reveals that glycans attached to proteins contribute not only to their solubility or stability but also to the tertiary structure of functional sites in proteins. It is becoming more and more important to elucidate the role of glycans from the structural aspects in the basic life science and applied science field, such as drug development. In this situation, we have been developing a methodology for analyzing the structure of glycoconjugates by nuclear magnetic resonance (NMR) spectroscopy. In particular, we have established a systematic method for stable isotope labeling of glycoproteins with a variety of techniques based on synthetic chemistry, enzyme chemistry and cell cultivating. Hence, it is possible to analyze the structure, dynamics and interaction of glycoproteins at atomic resolution.