New Approaches And Materials For The Enrichment And Separation Of Cis-diol-containing Biomolecules

Biomolecules containing one or more cis-diol groups,such as glycoproteins,nucleosides and carbohydrates,can be called cis-diol-containing biomolecules(CDCBs).They play vital roles in bio-systems and are important target compounds in x-omics analysis.Because CDCBs are present in a large number of isomers or structural analogues,and their abundances in complex biological samples are usually very low and interfered with the high abundance components.Selective enrichment and efficient separation of the target CDCBs are two key aspects in the analysis of these compounds.In this paper,we focused on new approaches and materials for the enrichment and separation of CDCBs.Erythropoietin(EPO)is an important glycoprotein hormone.Recombinant human EPO(rhEPO)is an important therapeutic drug and can be also used as doping in sports.However,due to the presence of a large number of glycoprotein isoforms with small differences in the glycan structures,the trace concentration in urine and blood,and the complexity of sample matrix,high-resolution separation,sensitive detection and effective and facile sample preparation are essential issues.We explored new possibilities for these aspects.An immunomagnetic beads-based extraction(IMBE)method was developed to specifically extract EPO glycoforms;capillary zone electrophoresis(CZE)allows high resolution glycoform profiling for EPO while deep UV laser-induced fluorescence detection(deep UV-LIF)was established to detect the intrinsic fluorescence of EPO.By combining IMBE with CZE-deep UV-LIF,the overall detection sensitivity was enhanced by two orders of magnitude relative to conventional CZE with UV absorbance detection.Moreover,the whole process is simple,rapid and labeling-free.It is applicable to the analysis of pharmaceutical preparations of EPO.However,for anti-doping analysis,the sensitivity needs further improvement.CDCBs are usually highly hydrophilic compounds and are therefore difficult to achieve satisfactory separations using conventional reversed-phase-based micellar electrokinetic chromatography(MEKC)due to poor retention.We develop a new method,called boronate affinity-assisted micellar electrokinetic chromatography(BAA-MEKC),to solve this issue.The principle is as follows:a boronic acid with a hydrophobic alkyl chain is added to the background electrolyte and acts as a modifier to adjust the selectivity.CDCBs can covalently react with the boronic acid to form negatively charged surfactant-like complexes,which can partition into micelles formed with a cationic surfactant.Thus,CDCBs can be separated according to the differential partition constants of their boronic acid complexes between the micellar phase and the surrounding aqueous phase.To verify this method,eight nucleosides were employed as the test compounds and their separation confirmed that the combination of boronate affinity interaction with MEKC can effectively enhance the separation of CDCBs.Finally,the BAA-MEKC method was applied to the separation of CDCBs extracted from human urine,the results demonstrated that BAA-MEKC exhibited better selectivity as compared with conventional MEKC and CZE.Successful quantitative analysis of urinary nucleosides by BAA-MEKC was demonstrated.Boronic acids can covalently interact with CDCBs to form stable cyclic esters in an alkaline aqueous solution while the boronate esters dissociate when the environmental pH is switched to acidic.The property of pH-dependent switch makes boronic acids attractive affinity ligands in important applications ranging from chemical or biological sensing and separation to drug delivery.Boronic acid-functionalized materials,such as macroporous monoliths,magnetic nanoparticles and mesoporous materials,have gained increasing attention in proteomic analysis in recent years,due to their capability to selectively extract glycoproteins.However,boronic acids are associated with an apparent limitation,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,while many glycoproteins with important research or diagnostic value usually exist in very low abundance in biological samples.Therefore,it is an essential to develop boronic acid-functionalized materials to significantly improve the affinity of boronic acid-functionalized materials towards glycoproteins.Molecular recognition in biological systems provides an important clue,biomolecules,such as antibodies,can strongly bind their target molecules because of the combined synergistic strength of multiple binding interactions.To increase the avidity towards glycoproteins,dendrimeric boronic acid-functionalized magnetic nanoparticles(dBA-MNPs)were designed and synthesized.The boronate avidity material can bind glycoprotein through synergistic multivalent binding between multiple boronic acids on the dBA-MNPs and multiple sugars of glycoprotein,exhibit dissociation constants of 10-5-10-6 M towards glycoproteins,which are 3-4 orders of magnitudes higher than the binding in which no synergistic binding was involved.Due to the significantly improved binding strength,the boronate avidity magnetic nanoparticles not only allowed for efficient enrichment of glycoproteins of low concentration but also were tolerant for the presence of abundant competing sugars.Besides,binding capacity and binding/desorption speed were also enhanced.Such a strategy is generally applicable and can be applied to other types of boronic acid-functionalized materials and other types of functionalized materials.