Glycosylation is the one of the most challenging post-translational modifications. It usually has a set of modifications instead of a single modification. Even a single glycosylated site on a peptide may have a number of glycan isoforms with different chain length and branches attached. O-glycans attach to a polypeptide mainly through a glycosidic bond between the terminal monosaccharide residue and the OH group of Serine or Threonine. Unlike N-glycans, there is no known O-linked amino acid consensus sequence yet. The O-glycans are structurally diverse. They can be divided into several structural families with relatively heterogeneous core structures.
Figure 1. Different core structures of N-linked glycans
So far no universal enzyme can remove the majority of O-linked glycans, unlike N-glycans. The O-glycanase only removes unsubstituted disaccharide O-GalNAc initiated glycans. Chemical release is usually the method of choice for O-glycan release. The O-glycans are released from the O-glycopeptides mainly by β-elimination.
Figure 2. The general procedure of sample preparation for Glycan profiling
O-glycans profiling is usually performed using MALDI-TOF MS. It is necessary to derivatize O-glycan by permethylation to transform all hydroxyl groups into methyl ethers and stabilizes sialic acids by methyl esterification of their carboxyl groups. This allows MALDI TOF MS to analyze of all O-glycans in the positive mode. Direct infusion of O-glycans of permethylation into the mass spectrometer can provide information about glycans composition.
Figure 3. Example of O-Glycan structures detected and analyzed by using MS1
1. S. Yang, Y. Hu, L. Sokoll & H. Zhang (2017). Simultaneous quantification of N- and O-glycans using a solid-phase method, Nature Protocols, 12, 1229–1244.
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