Overview of n-glycan

Posted by sherrycryer on October 13th, 2018

Glycosylation (also chemical glycosylation) is the one of the most challenging enzymatic processes which attaches glycans to proteins or other organic molecules, it is a form of co-translational and post-translational modification. This enzymatic process will produce one of the fundamental biopolymers found in cells. To be specific, even a single glycosylated site on a peptide may have a large number of glycan isoforms with different chain length and branches attached.

Glycans serve a variety of structural and functional roles in a membrane and secreted proteins. For instance, the majority of proteins synthesized in the rough endoplasmic reticulum undergo glycosylation and it is an enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of glycation. Glycosylation is also present in the cytoplasm and nucleus as the O-GlcNAc modification. However, there is difference and similarities between O-glycans and N-glycans.  When it comes to similarities, they both are both are typically found in proteins processed through the ER and Golgi. But they are different in some area. For example, O-glycansattach to a polypeptide mainly through a glycosidic bond between the terminal monosaccharide residue and the OH group of Serine or Threonine while there is no known O-linked amino acid consensus sequence and N-glycans are attached to the amino side chain of an asparagine residue. Besides, The O-glycans are structurally diverse as they can be divided into several structural families with relatively heterogeneous core structures. So far scientists have not found any universal enzyme can remove the majority of O-linked glycans, unlike N-glycans. The O-glycanase only removes unsubstituted disaccharide O-GalNAc initiated glycans. The method of choice for O-glycan release is normally Chemical release. The O-glycans are released from the O-glycopeptides mainly by β-elimination. The final product of the n-glycan synthesis contains one of three possible types of glycans: Oligomannose, Complex and Hybrid and these can be further modified with additional sugars, while O-glycans can have 8 core structures, and when they get elongated, they are quite smaller.

Therefore, it is critical to identify and quantify both N- and O-linked glycans of glycoproteins to determine and identify the changes of glycans. 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. We achieve O-glycans profiling by three steps, including Sample preparation, released method, glycan label, Analysis of permethylated N-glycans by MALDI-TOF TOF. Firstly, we provide excellent sample preparation before deglycosylation step and then the exchange of buffer, concentration, enrichment or purification of the protein if necessary, thus the O- glycans can be released from glycoproteins or glycopeptides. Secondly, O-glycans are released from the O-glycopeptides or O glycoproteins by β-elimination. Thirdly, O-glycan profiling of permethylated O-glycans is performed using MALDI-TOF MS. Permethylation transforms all hydroxyl groups into methyl ethers and stabilizes sialic acids by methyl esterification of their carboxyl groups. Last but not least, we offer Analysis of permethylated N-glycans by MALDI-TOF TOF.