Development Of New Enrichment And Analytical Method For N-glycosylation Proteins, Peptides And Glycans Based On New Materials

Protein N-glycosylation is an important post-translational modification.N-glycosylation is involved in almost every important life process, such as fertilization,growth, immune response, cell recognition and communication etc. Many researches haveindicated that protein modification of N-glycosylation exists in the emergence anddevelopment of most severe diseases, for example, diabetes, heart failure, nervous systemdiseases and malignant tumor. Therefore a research on N-glycosylation is meaningful tomany biological processes as well as clinical research on malignant tumor. However,glycoprotein is low-abundance protein; glycan lacks functional groups; the stronghydrophilicity and slight heterogeneity of glycan make it a challenge to analyze proteinglycosylation. In order to analyze protein N-glycosylation with high throughput, highsensitivity and on large scale, a convenient, fast and specific enriching method ofN-glycoproteins, glycopeptides and glycan is urgently needed.This thesis is based on the above issues and mainly discusses the novel methods onhighly efficient separation and enrichment of N-glycoprotein, glycopeptide and glycan. Wehave done pioneering work in synthesizing graphene, hydrazide functional temperaturesensitive polymer and pH sensitive polymer. These compounds have been successfullyapplied in the enrichment and detection of N-glycan and glycopeptide of human plasma,mouse livers and mouse brain proteins. The scale of protein glycosylation beingidentificated is obviously improved.The first chapter introduces the research status of glyco-proteome including theseparation and enrichment technique of N-glycoproteins, glycopeptides and glycan. Theapplication of hydrazine in identification of protein glycosylation is mainly discussed.The second chapter introduces a processing method of enriching and derivingN-glycan within one step based on graphene.Graphene reacts with aromatic compounds byπ-π conjugation. Meanwhile, hydrazide on aromatic compounds can react with hemiacetalon N-glycan by covalent coupled reaction. We innovatively complete the enrichment,derivation and desalting within one step. The procedure is simplified, the throughput is enhanced and the loss is reduced. This one-step-method was used in maltoheptaoseprocessing and the sensitivity was improved by33folds in MALDI-TOF MS. Moreover,48N-glycoforms were successfully identified in human plasma by using this method.In the third chapter, the hydrazide functional temperature sensitive polymer wassynthesized by radical polymerization of isopropylacrylamide and methyl acrylate, which isinduced by potassium persulfate. The states of polymers in solution can be controlled byadjusting the temperatures of the system. We could fix the temperature to dissolve thepolymers which resulting in high efficiency enrichment of N-glycoproteins andglycopeptides in homogeneous phase, while non-glycoprotein and non-glycopeptides willbe precipitated and eluted from the system. The homogeneous reaction greatly simplifiesthe exchanges of materials in the enriching process. Hence, the reaction time is reduced andthe enriching efficiency is enhanced. This polymer had been used in the enrichment ofN-glycopeptide in a mixed sample of bovine fetuin and bovine serum albumin (1:100, w/w)and mouse liver sample. The three N-glycopeptides of bovine fetuin were successfullybeing enriched and identified. The disruptions of non-glycopeptides, which have higherresponse in mass spectrometry, were wiped out.466N-glycopeptides were identified bythis method in mouse liver samples, corresponding to213N-glycoproteins.In the fourth part, we synthesize hydrazide functional pH sensitive polymers by radicalpolymerization of acrylic acid and methyl acrylate, which is induced by potassiumpersulfate. The polymer is used in the enrichment of N-glycopeptides in a mixed sample ofbovine fetuin and bovine serum albumin (1:100, w/w) and mouse brain samples. Thepolymers could dissolve completely by controlling the pH of system in the enrichingprocess, thus the enriching efficiency is enhanced greatly. The three N-glycopeptides hadbeen successfully enriched in the mixed sample and the disruption of non-glycopeptideshad been wiped out.1276N-glycopeptides were identified by this method in mouse brainsamples, corresponding to442N-glycoproteins. The enrichment efficiency was furthercalculated as65%according to the ratio of the number of the identified N-glycopeptide andthe total peptide. This indicates that both the hydrazide functional temperature sensitivepolymers and pH sensitive polymers are highly efficient and specific to N-glycopeptideenrichment and have a good application prosepect in glyco-proteome.