Mass spectrometry-based structure-specific <?A3B2 pi6?><italic>N-</italic>glycoproteomics and biomedical applications
Ming Bi, Zhixin Tian
Abstract
<p indent="0mm"> <italic>N-</italic>linked glycosylation is a common posttranslational modification of proteins that results in macroheterogeneity of the modification site. However, unlike simpler modifications, <italic>N-</italic>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 <italic>N-</italic>glycosylation, i.e., the same <italic>N-</italic>glycosylation site can be modified with different glycans with a certain stoichiometric ratio. <italic>N-</italic>glycosylation regulates the structure and function of <italic>N-</italic>glycoproteins in a site- and structure-specific manner, and differential expression of <italic>N-</italic>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 <italic>N-</italic>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 <italic>N-</italic>glycopeptides and the clinical significance of differentially expressed intact <italic>N-</italic>glycopeptides. With the help of these modern methods, structure-specific <italic>N-</italic>glycoproteomics has become an important tool with extensive applications in the biomedical field.