Establishment of labelling methods for the analysis of glycan biomarkers of pancreatic cancer

Abstract

Glycoproteins play an important role in many biological processes at the cellular level, such as recognition, signalling and adhesion to other molecules. Many of these functions are controlled and modulated by the oligosaccharides, also known as glycans, attached to these proteins. Moreover, glycan structure can be altered in several diseases such as congenital disorders, chronic inflammation or cancer. Hence, detection and characterization of these glycans has become an interest target in this field with the purpose of establishing concrete glycan-biomarkers for each of the mentioned diseases. Human alpha acid glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in pancreatic cancer (PDAC) and chronic pancreatitis (ChrP). Therefore, some of its glycans are considered potential biomarkers for these diseases and many recent studies have focused on the research of a sensitive and reproducible method for their labelling. Among glycan derivatization methods, reductive amination followed by glycan separation by high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) both coupled to mass spectrometry detection (MS) is one of the most widely used. In the present work, four different labels: anthranilic acid (2-AA), aniline (AN), procainamide (ProA) and 8-aminopyrene-1,3,6-trisulfonic acid (APTS), were evaluated using maltohexaose (MH) as standard glycan. The main purpose of this study was to optimize a derivatization method for each considered labelling reagent. Once the optimal conditions were found, the selected methods were used to derivatize hAGP N-glycans after their release from the glycoprotein. The labelled glycans were analysed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and capillary zwitterionic hydrophilic interaction liquid chromatography electrospray ionization time-of-flight mass spectrometry (capZIC-HILIC-ESITOF- MS) in order to compare sensitivity and reproducibility in complex type glycans