Separation and characterization of α2-3 and α2-6 isomeric sialylated 0-glycopeptides from proteolytically digested caseinomacropeptide using hydrophilic interaction liquid chromatography (HILIC) - tandem mass spectrometry

Abstract

Most carbohydrates can be attached to proteins by two different bonds, called N- and O- linkages. N-linked glycosylation requires a specific amino acidic sequence whilst O-linked glycosylation consists of attaching the glycans to the hydroxyl oxygen of any serine or threonine residue. Generally, O-glycans are attached to the protein through N-acetylgalactosamine and sialic acid (Neu5Ac) is found to be terminating branches of glycans. The most common linkages found in the sialic acid are to the C-3 or C-6 positions of galactose residues. Bovine caseinomacropeptide, a fragment derived from the action of chymosin or pepsin cleavage of the kappa-casein, has two previously characterized isomeric trisaccharides, containing the α 2-3- and the alpha 2-6- linked sialic acid, respectively. Currently, hydrophilic interaction liquid chromatography (HILIC) is gaining importance in the analysis of glycopeptides, but little information is found about O-glycan and O-glycopeptide analysis. Therefore, in this work a HILIC multi-stage mass spectrometric method (HILIC-ESI-MSn) has been developed to characterize α 2-3 and α 2-6 sialylated trisaccharides either in their unconjugated form or linked to the bovine CMP. Proteolytically digested CMP and a tetrapeptide model non-enzymatically glycosylated with two different sialyl-trisaccharides were employed. The separation was carried out on a zwitterionic HILIC column, using a linear gradient of acetonitrile and water, with 0.005% v/v of formic acid, coupled to an ion trap mass spectrometer at positive and negative modes. CMP glycopeptides differing only in the isomeric sialylated glycan structure, i.e. linear (α 2-3) and branched (α 2-6) depending on the attachment of the sialic acid, could be separated by a ZIC®-HILIC column under the chromatographic conditions described above. Furthermore, this same behavior was also found in the tetrapeptide model glycosylated non-enzymatically with two isomeric sialyl-trisaccharides. Knowing the studied trisaccharide structures, the values of (w^w)pKa and (w^s)pKa were calculated. The branched trisaccharide was less acidic (higher pKa values) than the linear trisaccharide. Consequently, it could be expected that glycopeptides with the branched trisaccharide had a higher retention time due to a low degree of electrostatic repulsion interactions between the negatively charged sialic acid residue and the negatively charged terminal sulfonate group of the stationary phase than their counterparts with a linear isomeric trisaccharide. This behaviour was confirmed by the MS2 detection of the fragment Y1β-type ion corresponding to the initial neutral loss of Gal residue, denoting the presence of this carbohydrate at the terminal position of the glycan structure and, therefore, revealing the presence of the branched trisaccharide. In conclusion, ZIC-HILIC-ESI-MS2 has shown to be a powerful technique for the separation and identification of α2-3 and α2-6 isomeric sialylated O-glycopeptides without any derivatization treatment.