Development Of Microfluidic Chip Methodologies And Platforms For N-linked Glycosylation Analysis

Glycosylation plays an important role in biological and physiological processes such as antigenicity, secretion, clearance of glycoproteins and virus invasion. Research in glycosylation may contribute to the development of proteomics and drug screening. This thesis focuses on the development of new methodologies and platforms based on microfluidic devices and/or mass spectrometry (MS) for the analysis of protein glycosylation.Mono- and oligo- saccharides were separated by microchip electrophoresis with direct and indirect laser induced fluorescence (LIF) detection. The whole separation process takes a few minutes. Systematically, a new method was developed for the glycoprotein analysis with microchip electrophoresis combined with exo-glycosidase digestion. Glycopeptides of turkey ovalbumin were found to contain high-mannose and hybrid type oligosaccharides using this method. Furthermore, a lectin affinity microfluidic chip was designed and fabricated for glycoform separation. A micro-scale monolith-based lectin column was developed. Using immobilized pisum sativum agglutinin (PSA) column on a chip, glycoforms of glycoproteins, chicken ovalbumin, turkey ovalbumin and ovomucoid of different glycan structure were fractionated into non-bound, weakly bound and strongly bound fractions according to the type of glycans they carry respectively. The integrated lectin affinity chromatography on a microfluidic chip resulted in a significant reduction in the analysis time and sample consumption. In addition, a stable and sensitive sheath flow nano electrospray interface wasdeveloped to couple glass microchip with mass spectrometry. The microchip/MS system was used as a platform for glycosylation analysis. Glycoforms and glycopeptides from glycoprotein were analyzed using microchip electrophoresis- quadrupole ion trap MS. In addition, the compositions of glycans of intact glycoproteins were determined using MALDI-TOF-MS with exo-glycosidase digestion. The structure of glycans was further analyzed with MS/MS measurement on MALDI-Q-TOF-MS.