Preparation Of Cyclodextrin-based Monolith And Application In Glycoproteomics

Glycosylation is a post-translational modification involved in numerous human diseases of widespread impact.Analysis of glycopeptides is therefore essential in the search of new biomarkers to gain a better understanding of the causes and progression of diseases,and in the monitor of responses to clinical treatment.Mass spectrometry(MS)is indispensable in glycosylation analysis,but it is still far from routine due to low abundance of glycopeptides and heterogeneity of glycan structures.Therefore,an enrichment step of glycoproteins or glycopeptides is critical before MS analysis.Organic polymer monolith column has been recently developed as a new separation media,which is fabricated with unsaturated functional monomers and cross-linkers inside an activated capillary and anchored to the wall through chemical bonding by in situ polymerization.In recent years,PMME method in MS determination of glycoprotein or glycopeptides occupies an important place.Reported results show that PMME method take advandeges of strong extraction ability,organic solvent consumption,short time consuming,removing most of the impurities in the sample preparation step disturbance,and purification of target analytes.?-Cyclodextrin(?-CD),a well-known host molecule,contains a hydrophobic internal cavity and hydrophilic external surface.Due to their unique molecular structure,?-CD can be selectively combined with various organic,inorganic,and biological guest molecules.?-CD related glycosylation enrichment materials have attracted great attention to enrich glycopeptides from biosamples.The aim of the present study is to design and synthesize ?-CD functionalization monolith materials coupled with MS for the determination of glycopretins or glycopeptides.The following four sections are included.1.In the present work,we designed a new kind of glycosylation poly(hydroxyethyl methacrylate-pentaerythritol triacrylate)monolith functionalized with glycocluster grafted ?-cyclodextrin for the enrichment of glycoproteins.The introduced modifiers endowed the monolithic material with enhanced hydrophilicity and surface area,which benefitted to improve the enrichment selectivity and extraction efficiency for glycopeptides.By combining with MALDI-MS detections,22 glycopeptides from horseradish peroxidase digest were captured with the developed monolith.When applied to the enrichment of glycopeptides from complex protein samples and human lymphoma(U937)cell line,the prepared monolith exhibited high selectivity for glycopeptides.2.A facile online method coupling PMME with MS was developed for the detection of IgG galactosylation glycopeptides.A PNA-?-CD functionalized poly(hydroxyethyl methylacrylate-ethyleneglycol dimethacrylate)monolith was designed via a click reaction.Thanking to the specificity of PNA-?-CD for the targets,the material exhibited enhanced enrichment selectivity and extraction efficiency for IgG galactosylation glycopeptides.The detection limit of IgG galactosylation glycopeptides as low as 0.5 fmol was achieved.The PMME-MS method was applied to IgG galactosylation glycopeptides in real samples including human serum and acute myelogenous leukemia cell lysate.3.In the present work,Lys-based adsorbents were prepared for the specific capture of Plg through the covalent binding of Lys with a polymer monolithic substrate.Lys was modified with ?-CD via a click reaction and anchored to the substrate with biotin by host–guest interaction.The biotin-Lys-CD based monolithic material was employed for the specific capture of Plg.Combining with MS determinations,the method exhibited a low detection limit of 1.0 fmol with relative standard deviations below 10.0% for Plg.4.CDVs were firstly introduced into polymer monolith to prepare pH-responsive adsorption material and used for capture and release of a cardiac biomarker,myoglobin(Myo).SH-CDV was decorated with adamantane modified SH-octapeptide to enhance the encapsulation and release rates of Myo.Afterwards,SH-CDV was introduced into polymer monolith via click reaction to produce a pH-responsive monolith.Combining with mass spectrometry detection,the CDV-based p H-responsive monolith was used for enrichment of Myo glycopeptides from mixture of glycopeptides and nonglycoprotein(BSA)tryptsin digests reach up to 1:10000.A limit of detection of 0.1 fmol was obtained for Myo glycopeptides in blood sample,indicating the high sensitivity of the method.