Highly Selective Separation And Enrichment Of Glycopeptides And Phosphopeptides Based On Smart Polymers

Protein glycosylation is one of the most studied post-translational modifications due to its important roles in cell biology and diseases.In addition to the major O-and N-linked glycosylations,C-mannosylation,protein phosphoglycation and Glycosyl Phosphatidyl Inositol?GPI?anchor glycosylation?or glypiation?have been identified.Such protein modifications can considerably change the secondary structures of proteins,which may alter their activities or interactions with other molecules and consequently,affect diverse biological processes.Thus,aberrant glycosylation may be closely related to the progression of diseases such as cancer,neurodegenerative disorders and other genetic abnormalities.Therefore,to fully exploit the potential applications of glycoproteins in prognosis and diagnosis,it is not only essential to detect the glycoprotein biomarker or to analyze differences in disease-associated glycoforms in order to differentiate between normal and diseased states for biomarker discovery and drug development.Therefore,developing new methodologies to discern phenotype-dependent glycosylation will not only explain the mechanistic aspects of cell signaling cascades but also accelerate biomarker discovery for disease diagnosis or prognosis.Protein glycosylation mainly focus on the determination of the peptide sequence and the glycosylation site as well as the characterization of glycan composition and the structure.However,it is still a great challenge for glycoproteomics to selectively separate and enrich the glycopeptides and phosphopeptides from the complex biological samples due to their extremely low concentrations.This dissertation paid much attention to this hot and difficult issue in this field,namely the separation and enrichment of glycopeptides and phosphopeptides.In the general,this dissertation was divided into two important parts.The first part described the glycopeptides enrichment and separation based on phenylboronic acid methods.It is well known that in the basic buffer solution phenylboronic acid?PBA?-containing materials can form reversible diester bonds with cis-diol-containing compounds and capture the glycopeptides containing cis-diol structures.Whereas in acid conditions the diester bonds broke,these PBA materials released the glycopeptides.Basing on this method,we synthesized and grafted the copolymer poly?PNI-co-TF-co-PBA?and poly?PNI-co-ATPBA?from SiO₂ spheres,which were then characterized by SEM,XPS,BET and TGA to make sure the copolymers were successfully coated on silicon surface.Subsequently,we thoroughly evaluated the separation and enrichment efficiency of these materials towards glycopeptides and phosphopeptides in different conditions.The second part illustrated the separation and enrichment of glycopeptides based short peptides,including the fourth and fifth chapter of this dissertation.Inspired from the selective recognition of lectin to diverse saccharides lied in the key short peptides in the structure of lectin,we designed and developed a series of three-componentcopolymersincludingpoly?PNI-co-PT-co-DF?,poly?PNI-co-?-Asp-Phe-co-PT?and poly?PNI-co-?-Asp-Phe-COOH-co-PT?,which were comprised of dipeptide-based recognition units,phenylthiourea-based mediating units and a flexible poly?N-isopropylacylamide?backbone as the functional switching unit.Firstly,the binding capacities of the dipeptides or dipeptides-phenylthiourea towards various kinds of saccharides were investigated through fluorescent titration experiment.Then,these functional monomers were grafted from Au resonators and silicon wafer to monitor the adsorption of diverse saccharides through a quartz crystal microbalance and study the macroscopic properties changes of materials?i.e.surface wettability and adhesion force?with AFM,after the polymers modified silicon wafer being treated with saccharide solutions.Subsequently,these polymers were grafted from SiO₂ spheres through ATRP,which were then thoroughly characterised by SEM,BET,XPS and TGA to guarantee the polymers were successfully grafted to the surface of SiO₂ spheres.Finally,we systematically evaluate the separation and enrichment efficiency of these materials towards glycopeptides as well as phosphopeptides in different conditions.Insummary,throughsynergetichydrogenbondinginteraction,the three-component copolymer materials exhibited distinct adsorption behaviors,surface wettability and adhesion force transition towards different disaccharides.Furthermore,as a typical example,we successfully applied these smart features in glycopeptide and phosphopeptides enrichment and established a relatively well-developed evaluation system.This provides much enlightenment for the applications of smart polymers in saccharide separation and proteomics.