Preparation Of Functionalized Two-dimensional Metal-organic Frameworks For Application In The Separation And Enrichment Of Glycopeptides

Protein glycosylation,one of the most prevalent post-translational modifications of proteins,plays an important role in regulating many complex biological processes.Due to the low abundance of glycopeptides in biological samples and their susceptibility to interference during mass spectrometry,it is essential to separate and enrich glycopeptides prior to analytical detection.Metal-organic frameworks(MOFs),as a porous material with large specific surface area,have been widely used in the field of separation and enrichment.However,the MOFs used for glycopeptide enrichment are often threedimensional in structure,which have large spatial resistance and limited binding sites in glycopeptide enrichment,resulting in low glycopeptide enrichment efficiency.Based on the above problems,this work took the advantages of twodimensional MOFs in glycopeptide enrichment and enhances the glycopeptide enrichment performance of two-dimensional MOFs by post-synthesis modification.The main research contents are as follows:(1)A two-dimensional metal-organic framework composite(Zr-Fc MOF@Au@GC)functionalized with dual amino acids was constructed for the isolation and enrichment study of glycopeptides based on the hydrophilic interaction.The highly hydrophilic Zr-Fc MOF@Au@GC composites were obtained by in situ reduction of Au nanoparticles loaded on the surface of hydrophilic Zr-Fc MOF nanosheets,followed by modification of GSH and LCys by Au-S interactions.The two-dimensional Zr-Fc MOF had a smaller steric hindrance and more binding sites,while the surface modified GSH and L-Cys had a synergistic effect,making the composites exhibit excellent glycopeptide enrichment properties.The composites were used for the separation and enrichment of glycopeptides in HRP and Ig G digests,and 39 and 44 glycopeptides were detected,respectively.In the meantime,the composites exhibited ultra-high sensitivity(0.1 fmol/μL),excellent selectivity(HRP/BSA= 1:2000,w/w),satisfactory binding capacity(200 mg/g),and were still detectable after 5 times of repeated use 38 glycopeptides were detected in the HRP digest.In addition,the composite was further used in the glycopeptide analysis of real samples,and 655 glycopeptides corresponding to 366 glycoproteins were detected from human serum samples.These results indicated that Zr-Fc MOF@Au@GC exhibited excellent enrichment ability in both model proteins and actual samples,providing more information for indepth study of protein glycosylation in complex biological samples.(2)A benzeneboronic acid functionalized two-dimensional metal-organic backbone composite(Co/Fe-MOFs@Au@4-MPBA)was constructed for the isolation and enrichment study of glycopeptides based on the boronic acid affinity method.Co/Fe-MOFs@Au@4-MPBA composites with high loading of boronic acid groups were prepared by in situ loading of Au nanoparticles on the surface of 2D Co/Fe-MOFs,followed by modification of 4-mercaptophenylboronic acid(4-MPBA)through Au-S interaction.The composites combined the smaller steric hindrance and more binding sites of 2D MOFs with the higher selectivity and specificity of the boronic acid affinity method for glycopeptides,and enriched 34 and 35 glycopeptides in HRP and Ig G enzymatic digests,respectively.Meanwhile,when the composite material was enriched for glycopeptides in HRP digest,18 glycopeptides were still enriched when the ratio of glycopeptides to interfering protein BSA reached1:2000;3 glycopeptides could still be detected in glycopeptide concentrations as low as 0.1 fmol/μL;and the number of enriched glycopeptides remained basically the same after 6 times of repeated use.These results revealed that Co/Fe-MOFs@Au@4-MPBA have excellent glycopeptide enrichment performance and were expected to be used for the study of protein glycosylation in real biological samples.