A New Method Of Enrichment And Identification Of Glycopeptide Based On Functionalized Nanomaterials And Biomass Masses

This thesis presents an interdisciplinary research involved in analytical chemistry, chemical biology, materials chemistry as well as relevant biochemical techniques. Based on the biological mass spectrometry (MS) and a series of newly-designed magnetic nano-composites and polymer nano-materials, the high efficient and high sensitivity enrichment and identification methods have been developed and successfully used in analysis of complex samples such as human serum.As one of the most important and universal protein post-translational and modifications (PTMs), glycosylation is widely existed in more than 50%of all proteins. It plays a significant role in differentiation, development, the metastasis of tumor, immunity, infection and regeneration. It is also closely related to the development of some diseases, such as the early diagnosis of tumor, process monitoring and prognosis evaluation. Therefore, the research on glycosylation has special biological significance and clinical value and has always been the hotspot in the field of proteomics. However, due to the low abundance of glycoproteins, it’s difficult to obtain enough quantity of the samples to further analysis unless the application of pre-enrichment. What’s more, there are enormous number of proteins and extremely wide dynamic range in the very complex biological samples, it’s really a challenge to separate glycoproteins from others. Therefore, the high abundant nonglycosylated proteins would suppress the identification of low abundance glycoproteins seriously. Moreover, glycosylation has the unique characteristic of microheterogeneity which leads to the vast diversity of glycan structures and the difficult determination of glycosylation sites.In MS analysis of glycosylation, we usually adopt the "bottom up" method which means studying it on peptide level. However, the abundance of glycopeptides accountes for only 2-5% in total when glycoprotein is digested into peptides. And the MS signals of nonglycosylated peptides always heavily interfere with those of glycopeptides. As a consequence, the pre-enrichment of glycoproteins/glycopeptides from complex biological samples with high selectivity and high sensitivity is of primary importance.Nowadays, continuous efforts have been made to develop strategies for separation of glycoproteins/glycopeptides from complex biological samples, but any of them are far from perfect. For example, boronic-acid based enrichment method is unbiased to both N-and O-glycoproteins/glycopeptides and facile to handle but has poor specificity. While hydrazide chemistry method owns the best performance of specificity but the poor performance in sensitivity and reaction time, the hydrazide materials are also expensive. Furthermore, the contaminants which were brought during the pretreatment would interference with MS analysis so that desalting or washing is needed. This process would cause loss of samples inevitably. In this thesis, we aimed to develop novel methods to address the above mentioned challsenges.In the first chapter, we give a brief introduction of glycosylation, the proceedings and challenges of glycolproteomics and the application of nano-materials in glycoproteins/glycopeptides enrichment. All of the background information provides theoretical and practical support on our research.In the second chapter, we introduce a new method for highly efficient and specific enrichment of glycopeptides using two different nanomaterials synergistically. It’s the first time to use two different nanomaterials for enriching glycopeptides in glycoproteomics field. One is boronic-acid functionalized nanoparticles Fe3O4@SiO2-APB to enrich glycopeptides, the other is conventional poly(methyl methacrylate)(PMMA) nanobeads which have strong adsorption towards non-glycopeptides. By optimizing the proportion of these two materials, extremely high sensitivity and selectivity are achieved. Since the washing step is not necessary for these conditions, the enrichment process is simplified and the recovery efficiency of target glycopeptides reaches 90%. The superiority of this technique also comes from the good dispersibility and high specific surface area of nanoparticles and easy separation by external magnetic field. Of course, we applied it successfully into characterization of glycoproteome of human serum samples. Finally,147 different N-glycosylation peptides within 66 unique glycoproteins are identified in the sample volume as little as 1μL.In the third chapter, aniline functionalized magnetic nanoparticles Fe3O4@SiO2@Aniline has been successfully synthesized for the isolation of glycopeptides. Due to the unsurpassable specificity of solid phase extraction by hydrazide chemistry, we have been inspired by its mechanism and develop a novel technique based on the conjugation of aldehydes from oxidized glycopeptides and aniline group functionalized on the magnetic nanoparticles via non-reductive amination reaction. Compared with the hydrazide chemistry method, desalting is no longer needed which facilitates the realization of high sensitivity. Through the formation of stable Schiff base or glycosylamine, the reducing regent is also eliminated in this method. The whole experiment can be completed in just 2 hours. In addition, aniline functionalized nanomaterial is easily prepared and cheap. At last, we use this technique to map as much as 80 N-linked glycoproteins in only 5μL human serum sample.In summary, these two brand-new protocols have shed new light upon the study of glycoproteomics.