Mass spectrometry-based structure-specific glycoproteomics and biomedical applications.

linked glycosylation is a common posttranslational modification of proteins that results in macroheterogeneity of the modification site. However, unlike simpler modifications, glycosylation introduces an additional layer of complexity with tens of thousands of possible structures arising from various dimensions, including different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformations. This results in additional microheterogeneity of the modification site of glycosylation, i.e., the same glycosylation site can be modified with different glycans with a certain stoichiometric ratio. glycosylation regulates the structure and function of glycoproteins in a site- and structure-specific manner, and differential expression of glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis. Numerous advanced methods ranging from sample preparation to mass spectrum analysis have been developed to distinguish N-glycan structures. Chemical derivatization of monosaccharides, online liquid chromatography separation and ion mobility spectrometry enable the physical differentiation of samples. Tandem mass spectrometry further analyzes the macro/microheterogeneity of intact glycopeptides through the analysis of fragment ions. Moreover, the development of search engines and AI-based software has enhanced our understanding of the dissociation patterns of intact glycopeptides and the clinical significance of differentially expressed intact glycopeptides. With the help of these modern methods, structure-specific glycoproteomics has become an important tool with extensive applications in the biomedical field.