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

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

Type: Doctoral Thesis
Title: Rapid and sensitive methods for the analysis and identification of O-glycans from glycoproteins
Author: Kozak, R.P.
Issue Date: 2017-01-24
Keywords: O-linked glycans
Hydrazinolysis
O-glycan peeling
Glycan release
Procainamide
HILIC–(U)HPLC
ESI–MS
Saliva
Abstract: 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.
Promotor: Supervisor: A.M. Deelder, M. Wuhrer Co-Supervisor: D.L. Fernandes
Faculty: Faculty of Medicine, Leiden University Medical Center (LUMC)
University: Leiden
Uri: urn:isbn:9789462955660
Handle: http://hdl.handle.net/1887/45434
 

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