Rapid and sensitive methods for the analysis and identification of O-glycans from glycoproteins

摘要

Changes in both N- and O-glycosylation have been associated with many states of health and disease, providing prognostic and diagnostic information. O-glycans have been relatively little studied through lack of suitable analytical tools but the evidence is that they could be promising biomarkers.The release and analysis of O-glycans remains very challenging. The main reason for this is that there is no enzyme available for universal O-glycan release. Therefore, chemical release is the most effective method. Unfortunately all of the O-glycan release methods show a side reaction known as “peeling”. Peeling is well known problem when performing the O-glycan release and often results in poor repeatability. The aim of the work described in this thesis was to develop or improve methods for the release of O-glycans with free reducing termini with high yield and limited degradation. The development of sample preparation techniques for O-glycan release (in their nonreduced form) by hydrazinolysis are described. The use of procainamide for fluorescent labelling of glycans followed by HILIC-UHPLC-ESI-MS/MS analysis is introduced. The application of the improved protocols for O-glycan release by hydrazinolysis combined with developed procainamide labelling system followed by HILIC-UHPLC-ESI-MS/MS analysis for evaluation of O-glycosylation changes in human saliva.