Preparation Of Functionlized Covalent Organic Frameworks Composites For The Isolation And Enrichment Of Glycopeptides

Protein glycosylation is one of the most complex and important post-translational modifications of proteins and can be used as biomarkers to detect many diseases.Due to the complex peptides produced after enzymatic cleavage and the low abundance of glycopeptides in the glycosylation analysis strategy,it is critical to separate and enrich glycopeptides before analysis and detection.Covalent organic frameworks（COFs）are new types of porous organic materials made entirely of organic monomers linked by strong covalent connections to light elements.COFs with high specific surface area,regular framework structures and high chemical stability have been rapidly developed for separation and enrichment.However,most COFs enrichment materials in the field of glycopeptide isolation and enrichment only use the force between the graft group and the glycopeptide,which ignore the force between the COFs themselves and the glycopeptide,resulting in insufficient enrichment.Therefore,it is particularly important to develop novel materials to improve glycopeptide enrichment efficiency.In this work,different functionalized COFs composites were obtained by modifying hydrophilic or boronic acid groups on hydrophilic COFs.The hydrophilic group of COFs itself was used to enrich glycopeptides,and the grafted hydrophilic or boronic acid groups were modified to further improve the enrichment ability.Different functionalized COFs composites were used for the enrichment of glycopeptides in actual samples with good results.The main research contents are as follows:（1）We constructed a glutathione functionalized dual-hydrophilicity magnetic covalent organic frameworks composite（Fe₃O₄@Tp BD@Au@GSH）for the isolation and enrichment of glycopeptides.Tp BD was coated onto Fe₃O₄nanoparticles by precipitation polymerization and used as a carrier for modification.We used the interaction between oxygen and nitrogen atoms in Tp BD and Au nanoparticles to load Au onto the surface of Tp BD.Finally,GSH was grafted onto Tp BD through the Au-S interaction,which further enhanced the surface hydrophilicity of the prepared material.The double hydrophilicity of Fe₃O₄@Tp BD@Au@GSH was attributed to the ligands in Tp BD and modified GSH.The composite material showed excellent performance in enriching glycopeptides from standard proteins because of the hydrophilic nature of the COFs themselves and the hydrophilic of GSH,with 21 and 36glycopeptides enriched from horseradish peroxidase（HRP）tryptic digests and immunoglobulin G from human serum（Ig G）tryptic digests,respectively.The prepared composite exhibited ultra-high sensitivity（0.1 fmol/μL）,excellent selectivity（HRP tryptic digest/bovine serum albumin（BSA）tryptic digest=1:2000）and macromolecular protein anti-interference ability（HRP tryptic digest/BSA=1:2000）.Moreover,Fe₃O₄@Tp BD@Au@GSH exhibited outstanding binding capacity（160 mg/g）,excellent long-term storage capacity and good recycling ability.Glycopeptide enrichment of biological samples by Fe₃O₄@Tp BD@Au@GSH was successful,with 492 and 160 glycopeptides,corresponding to 134 and 64 glycoproteins,respectively.The results showed that Fe₃O₄@Tp BD@Au@GSH had an excellent performance in the field of glycopeptide enrichment.It provided more information to facilitate in-depth analysis of glycopeptides in biological samples.（2）We constructed a hydrophilic and affinity bifunctional covalent organic frameworks composite（Tp Pa@Au@MPBA）modified with mercaptophenylboronic acid for the isolation and enrichment glycopeptides.The interaction between the hydroxyl and amino groups in the Tp Pa ligand and the Au nanoparticles was used to load Au onto the Tp Pa surface,and finally grafted MPBA onto it via Au-S interactions.The synthesized bifunctional composites（Tp Pa@Au@MPBA）exhibited hydrophilicity and affinity.The composite used two strategies for the simultaneous enrichment of glycopeptides,improving on the disadvantages of single using hydrophilic interactions or affinity interactions.The prepared material was used for the enrichment of glycopeptides from standard proteins,with 34 and 42 glycopeptides enriched from HRP and Ig G tryptic digests,respectively.The composite sensitivity was0.05 fmol/μL,selectivity was 1:2000,macromolecular protein anti-interference ability was 1:2000,binding capacity was 200 mg/g,and had good recycling ability.Glycopeptide enrichment of biological samples by Tp Pa@Au@MPBA was successful,with 530 and 185 glycopeptides,corresponding to 152 and 61glycoproteins,respectively.These results showed that Tp Pa@Au@MPBA had a good advantage in the field of glycopeptide enrichment,which had great potential for glycoproteomic analysis and early disease diagnosis in complex biological samples.