| Cis-diol compounds are class of biomolecules including nucleosides,glycoproteins,saccharides,and nucleotides,which play vital roles in various biological processes.Due to low abundances of these species in the complex biological samples,their identification and analysis is difficult.Therefore,specific capture and targeting of these biomolecules is important in a wide range of biological experiments and applications.Consequently,marvelous antibodies have been developed,but they are often restricted in availability,costly,and unstable in processing.Different modes of affinity chromatography have been in practice for selective and specific capture of these target biological compounds.Among them,hydrophilic interaction chromatography(HILIC)and boronate-affinity chromatography are widely applied due to their unique characteristics to selectively capture and release the target cis-diol compounds.In this work,these two affinity modes of chromatography are focused to selectively enrich the cis-diol small molecules,glycopeptides and glycoproteins.The content of this dissertation is organized as follow:(1)A selective approach to extract nucleosides,glycopeptides and glycoproteins using boronic acid functionalized GMA-MAA-DVB polymer is proposed.The novel polymer,reported for the first time in proteomics,have high BET surface area(132.8447 m2 g-1)which contribute to efficient enrichment and average pore size(20.3449 nm)to facilitates the nano confinement effect for strong interactions.Hydrophilic character of methacrylic acid and diethylenetriamine,along with inherent affinity of boronic acid for glycosylated biomolecules result in selectivity up to 1:500 for peptides and 1:1000 for glycoprotein.Binding constant for cis-diol compounds are in the range of 10-4 to 10-6 M and theoretical binding capacity upto 85 mg g-1 for HRP and 180 mg g-1 for IgG,respectively.Furthermore,sensitivity of boronic acid functionalized polymer(BFP)for glycoproteins and glycopeptides is 1 pg mL-1 and 0.04 ng mL-1(S/N?3),respectively.Finally,material is applied to enrich the glycoproteins from healthy human saliva sample and six glycoproteins are identified.(2)Boronic acid-based affinity materials often stuck to some serious issues like high binding pH and weak affinity,especially in the case of glycoproteins.Consequently,5boronoisophthlic acid is used as a novel affinity ligand for the selective capture and release of glycoproteins.Because the pKa value of 5-boronoisophthalic acid is 7.8 which is just closed to physiological pH and is ideally suitable for the fast binding and elution kinetics of glycoproteins to avoid their degradation and deactivation.5-boronoisophthalic acid is attached to the surface of polymer support using branched polyethyleneimine(PEI)which enhances the binding strength as it has multiple amine groups available for the attachment of 5-boronoisophthalic for synergistic interactions.The resulting affinity material is characterized and packed in a micropipette-tip using hydrophilic melamine foam as a frit to make the separation process smooth,simple,reliable,and robust.This boronic acid-based affinity tip exhibits binding constants for model glycoproteins in the range of 10-6-10-7 M,binding capacities in the range of 0.66 to 2 ?M g-1,and selectivity up to 1:1000(HRP to BSA)under optimized extraction conditions.Finally,the boronic-based affinity tip is successfully applied to selectively capture the glycoproteins from the human milk sample,especially lactoferrin which is highly important in dairy manufacture.3)Endogenous glycopeptides are significantly important in diverse pathological and physiological systems,but their direct analysis is severely hampered by their low abundance and presence of interfering species in biological fluids.MOFs are considered ideal candidates in this area,due to their perfect arrangements,three-dimensional structure and tunable porosity.Therefore,we synthesized the amine-functionalized titanium metal-organic frame-work(NH2MIL-125(Ti))by a simple hydrothermal method,characterized.Then,NH2-MIL-125(Ti)crystals were packed in pipette-tip using hydrophilic melamine foam,as supporting frit to make the process smooth,simple,reliable,and robust.Free amine groups,present in the structure imparted hydrophilicity and a unique pattern of porosity,contributing to the size exclusion effect that excluded the large-sized proteins up to 1:700(peptide to protein ratio).The affinity-tip showed a detection limit of 1 fmol ?L-1 and selectivity up to 1:1000(HRP digest to BSA digest).Moreover,repeatability and reusability were evaluated up to five rounds of enrichment using the same affinity tip,and scanning electron microscopic images revealed no structural changes in the MOF crystals.Finally,the MOF packed in pipette tip was applied to selectively capture the N-linked endogenous glycopeptides from a healthy saliva sample and 64 unique endogenous glycopeptides were identified.These results demonstrated the excellent potential of NH2-MIL-125(Ti)based affinity tip for glycopeptides which can be used to enrich trace glycopeptide biomarkers from the biological fluids.4)Micro-tip-based solid-phase microextraction is considered as one of the green and powerful analytical sample preparation techniques,but its efficiency is seriously affected by some basic issues such as tedious fabrication,instability of sorbent bed,and blocking of the tip,especially for biological samples due to low permeability.These issues were tackled by introducing a flexible and hierarchical substrate in the microtip,having good mechanical strength and specific functionality to capture the desired biomolecules.UiO-66-NH2-crystals were grafted hydrothermally on the surface of PDA coated elastic melamine foam(MF)sponge which not only maintained the good mechanical processing for in-tip packing but also accessed the MOF crystals on its surface.Considering the well-ordered and flexible structure of melamine foam,it was used as a substrate and for hydrophilic interaction liquid chromatography(HILIC).Metal-organic framework,due to its excellent characteristics,was grafted on its surface anchored by selfassembling of polydopamine.The resulting material was characterized and packed in the tip by just pressing the material in the conical structure of the tip.This affinity tip had good and tunable permeability and was used to selectively enrich glycopeptides as well as phosphopeptides.The affinity tip demonstrated excellent performance to enrich glycopeptides and phosphopeptides with a low limit of detection up to 0.5 fmol ?L-1 from tryptic digests of horseradish peroxidase and ?Casein,respectively,and was stable up to 5 rounds of enrichment.Moreover,this affinity-tip also exhibited high selectivity up to up to 1:1000(HRP digest to BSA digest)for glycopeptides and 1:200(?-Casein digest to BSA digest)for phosphopeptides and demonstrated several other fascinating characteristics such as;excellent size exclusion effect for the omission of large-sized proteins,modest backpressure,reproducibility,reusability,smooth enrichment and successfully applied to a human saliva sample.These promising strategies may be helpful for the analysis of cis-diol biomolecules for their selective recognition and isolation from complex physiological samples in order to explore pharmaceutical and biomedical analysis.In future,affinity tips with stable affinity sorbents and modest backpressure,could be coupled with robotic systems which may lead to the quantitative determination of nucleosides,glycans,glycopeptides or glycoproteins for their rapid analysis. |
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