| With the completion of the human genome project,proteomics,metabolomics and glycomics have been an important frontier and hotspot in the field of life science nowadays.Cis-diol-containing biomolecules of important biological meaning,such as glycopeptide,glycoproteins,nucleosides,saccharides,glycans,and so on,are important targets in glycoproteomics,metabolomics,and glycomics.All of them play essential roles in regulating biological events and disease early dignostics.However,they often exist in low abundance together with severe interferences.Thus,selective enrichment is the critical step prior to analysis of cis-diol-containing biomolecules.Due to the combination of boronate affinity and monolithic columns,boronate affinity monolithic columns have found important applications in analysis of cis-diol-containing biomolecules.Conventional boronate affinity chromatography required a basic working environment(usually pH? 8.5).However,the basic environment not only needs the tedious procedure of pH adjustment but also leads to the degradation of analytes.Lots of efforts have been made to overcome these issues in the past several years.Up to now,the working pH of the boronate affinity monolithic columns have been reduced to 5.0,covering pH range of most biological samples,such as siliva,tear and blood,but still not enough for all of the biological samples especially for urine(pH 4.5-8.0).Apart from the working pH,there are still several problems to need to be addressed in boronate affinity monolithic columns:1)weak binding affinity.The dissociation constants between boronic acids and sugars or glycoproteins range from10-1 to 10-3 M.Thus,boronic acid-functionalized materials usually fail to enrich glycoproteins of very low concentration;2)the relatively low capacity of most boronate affinity monolithic columns is not satisfied with the demand of modern high throughput analysis for biological samples.In addition to glycosalation,phosphorylation is another important post-translational modification in biological events.Protein phosphorylation plays a vital role in cellular signaling of numerous biological processes and abnormal phosphorylation is recognized as a cause of human disease.Mass spectrometry(MS)has been an indispensable tool for phosphorylation analysis.However,the mass spectrometric analysis of protein phosphorylation is still far from being routine,because it often suffers from low abundance of phosphopeptides,poor ionization efficiency,and signal suppression by abundant nonphosphopeptides.To solve these issues,enrichment of phosphopeptides is essential prior to MS analysis.Metal ion affinity chromatography had found important applications in analysis of protein phosphorylation.In recent years,metal-organic-frameworks(MOFs)with high specific surface area have also been used in selective enrichment of phosphopeptides.However,they often suffer from apparent disadvantages,such as tedious operation procedure,poor reproducibility.Most importantly,they failed in high-performance online nanoliter analysis.First,aiming at the exiting problems in binding pH(pH>4.5)of boronate affinity monolithic columns,we chose a boronic acid with very low pKa value towards cis-diols for the preparation of a novel boronate affinity monolithic column.The compound 3-pyridylboronic acid was chosen as the functional ligand because it exhibited not only a very low pKa value(4.4)but also high hydrophility.Besides,the organic-silica hybrid base monolithic capillary was used as a supporting material.As expected,the prepared 3-pyridylboronic acid-functionalized organic-silica hybrid monolithic capillary exhibited excellent performance towards cis-diols.The column exhibited an extremely low binding pH(4.5),which was the lowest as compared with boronic acid-functionalized materials reported in literature.Such a binding pH is highly desirable for direct application to urine samples.Due to the presence of zwitterionic pyridinium in the boronate ligand,the monolithic capillary showed enhanced affinity toward negatively charged cisdiol-containing compounds such as ribonucleotides;meanwhile it could function as an anion exchanger at acidic pH,providing secondary separation for negatively charged cis-diol-containing compounds.Second,aiming at the exiting problems in the low binding strength of boronate affinity materials towards cis-diols,we used branched polyethyleneimine(PEI)as a scaffold to amplify the number of boronic acid moieties.Using 2,4-difluoro-3-formyl-phenylboronic acid(DFFPBA)that exhibited ultrahigh affinity toward cis-diol-containing compounds as an affinity ligand,we designed and synthesized a branched boronic acid-functionalized monolithic capillary.The prepared column exhibited Kd values of 10-6-10-7 M toward glycoproteins and binding capacity of 37 ?mol/mL toward cis-diol-containing small molecules,was the highest among already reported boronate affinity organic monoliths.Besides,the boronate avidity monolithic column exhibited one additional beneficial feature,lowered binding pH(?6.5),1 pH unit lower than non-PEI assisted monolithic column.Third,aiming at the exiting problems in MOFs based affinity materials,we designed and synthesized a new type of MOFs cystal with high chemical and thermal stability.This amino groups functionalized MOFs(UiO-66-NH2)was then react with methacrylic anhydride to obtain the butyl methacrylate(UiO-66-NH-Met).Finally,UiO-66-NH-Met was copolymerized with crosslinker to form the MOFs-based monolithic column.The performance was demonstrated by the analysis of tryptic digeation of standard protein and nonfat milk. |
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