The conformations of glycans from glyconjugates present a great challenge both for experimental and theoretical methods. Their complexity is duo to the variety of possible monomeric units which are linked in a branched way and have differently populated conformational states. Assessing the configuration and position of glycosidic linkages of the N-glycan is helpful to better understand the complexity. Up to now, there are five common N-glycan linkages known. Among these five linkages, N-acetylglucosamine to asparagine (GlcNAcβ1-Asn) is the most common one. Other types of linkages to Asn include: glucose in laminin of both mammals and Archaea, N-acetylgalactosamine (GalNAc) in Archaea, rhamnosein bacteria.
For linkage position determination, N-glycans are released from glycoproteins by PNGase F cleavage. A stable substituent (an ether-linked methyl group) is then introduced onto each free hydroxyl group of the the released N-glycans. The glycosidic linkages are then cleaved by acid hydrolysis. The obtained individual methylated monosaccharides have free hydroxyl groups at the positions that were previously involved in a linkage. The general strategy involves reduction of the monosaccharides to produce alcohols at C-1, followed by derivatization of free hydroxyl groups. Individual components of the mixture of partially methylated, partially acetylated monosaccharide alditols can be detected by GC-MS.
Figure 1. Example of glycan linkage analysis
Several steps are involved in N-Glycan Linkage analysis:
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