Chemical deglycosylation of N,O-glycosylated proteins
Proteins that are glycosylated are difficult to sequence as the modification by the sugar groups can cause steric hindrance of the protease reducing cleavage efficiency, significantly change the mass of liberated peptides to be analyzed by mass spectrometry and make identification of modified peptides more challenging. Development of methods to chemically removal of O- and N-linked oligosaccarides, glycosaminoglycans and GPI-anchors from proteins without affecting polypeptide chain would be an important technological development for PCL.
To this end PCL has used a TFMS (trifluoromethanesulfonic acid) based protocol from Sigma that was adapted for treatment of a few micrograms of protein instead of milligrams with simultaneous improvement in preserving of the integrity of the polypeptide. This method is currently being applied to the analysis of glycosylation sites in soluble form of IL-15 receptor. In addition PCL will use this new approach for localization of protein glycosylation sites using Edman protein sequencing and mass spectrometry and determing the C-terminal sequences of GPI-anchored proteins.