New Methods For Analyzing Cis-diol Containing Biomolecules

Cis-diol containing biomolecules are of a special class of compounds among which many are the analytes of interest in the research frontiers of life science, such as glycoproteins and glycopeptides in proteomics, nucleosides in metabolomics, and saccharides in glycomics. There are two important scientific problems in the-omics analysis:first, many cis-diol molecules of biological importance are present in low abundance in the samples while interfering substances are usually present in high abundance, specific capture and effective enrichment of target cis-diol biomolecules becomes a key and challenging step; second, there is little difference between the same species of cis-diol containing biomolecules which makes the separation difficult and leads to the demonstration of high resolution separation methods. In view of these two problems, this dissertation is focused on developing new methods for analysing cis-diol containing biomolecules.1. Rapid and high-resolution glycoform profiling of recombinant human erythropoietin by capillary isoelectric focusing with whole column imaging detectionHuman erythroproetin (hEPO) is a glycoprotein hormone produced primarily by the kidney, which stimulates red blood cell production. Recombinant human erythropoietin (rhEPO), generally produced in Chinese hamster ovary (CHO) cells, can be used as not only a therapeutic protein but also a doping agent in sports. Profiling of EPO glycoforms is a critical means for quality control in pharmaceutical industrial and anti-doping analysis of misuse in sports. However, the existing methods for the analysis of EPO are associated with either time-consuming or poor resolution. A rapid and high-resolution glycoform profiling method was presented based on capillary isoelectric focusing (CIEF) with whole column imaging detection (WCID). Experimental conditions that influence the separation were investigated. Under optimized conditions, rhEPO from three different sources were resolved into distinct populations within5min with excellent reproducibility. The presented method exhibited the advantages of speed and high-resolution as compared with existing methods.2. Boronate functionalized magnetic nanoparticles and off-line hyphenation with capillary electrophoresis for specific extraction and analysis of biomolecules containing cis-diolsIn recent years, functionalized magnetic nanoparticles (MNPs) have drawn continuously increasing attention due to their great potential for capturing biological molecules or species. However, functionalized MNPs as nanoextraction probes and the coupling with a separation platform for chemical analysis have not extensively investigated yet. Boronate functionalized MNPs were synthesized and employed as extracting probes to capture and enrich cis-diol-containing biomolecules, and an off-line coupling method of the MNPs-based extraction with capillary electrophoresis (CE) was established by using pH junction, an on-line preconcentration technique in CE, as a bridge for the coupling. The prepared MNPs exhibited specific selectivity and sufficient capacity. The pH junction compressed a large injected sample volume into a much narrower sample zone and therefore significantly improved the detection sensitivity, solving the sensitivity mismatch between the MNPs-based extraction and CE. Experimental conditions for the pH junction and the desorption were optimized. Under the optimized conditions, the sensitivity was enhanced by42-fold as compared with regular CE. N,N-dimethylformamide was found to be an effective desorption promoter, which reduced the desorption time to a few minutes. With the established method, riboflavin in a human urine sample was determined.3. At-line coupling of magnetic-nanoparticle-based extraction with gel isoelectric focusing for protein analysisWe present a strategy for at-line coupling of magnetic nanoparticles-based extraction (MNE) with gel isoelectric focusing (IEF). The key to the at-line combination is to use anolyte or catholyte as the desorbing agent and the sample well as a nature adaptor. Thus any functionalized magnetic nanoparticles (MNPs) can be facily at-line coupled with gel IEF, provided that the extraction/desorption process is pH-controlled. MNPs extracted with target proteins are added into the sample well, which can function as a natural adapter. Once a focusing electric field is applied across the gel, proton ions migrating from the anolyte or hydroxide ions migrating from the catholyte can act as desorbing agent, releasing the proteins from the MNE probes. The released proteins are consequently focused into distinct bands where the local pH equals to their pI values. phenylboronic acid functionalized MNPs can release the extracted cis-diol containing biomolecules under acidic condition:Thus it is suitable for at-line coupling with gel-IEF for enrichement of glycoproteins and the profiling the glycoforms of the extracted glycoproteins. In addition to phenylboronic acid functionalized MNPs, MNPs with ion-exchange functionalities were also used in this study, in order to identified that all kinds of MNE probes, as long as the desorption is pH-controlled, can be coupled with IEF. The at-line combination exhibited several significant advantages, including selectivity, sensitivity and speed.4. Smart magnetic nanoparticles for specifically harvesting low molecular weight glycoproteinsLow-molecular-weight glycoproteins (LMW-GPs) are a treasure-trove for disease biomarkers discovery. We present magnetic nanoparticles (MNPs) with smart functions for specifically harvesting LWM-GPs. The smart MNPs are MNPs wrapped with a boronic acid-functionalized polymer network. Along with the basic benefit of conventional MNPs (magnetic separation), the smart MNPs can provide triple desired functions:1) size-restricting effect,2) specific affinity to glycoproteins,3) protection of the harvested LMW-GPs against degradation and contamination and4) fast extraction speed. The specific extraction is due to the specific affinity of the boronic acid ligand to the cis-diol moieties of glycoproteins. While the size-restricting effect, the protection function and extraction speed relay on the polymer network on the surface of the MNPs. The designed functions were experimentally verified. Threshold value of the size-restricting effect was found adjustable through changing the polymer chain length. The smart MNPs can be developed into promising nanoprobes for specifically harvesting not only LMW-GPs but also other cis-diol-containing biomolecules of biological importance, such as nucleosides and glycans.5. On-line coupling of Wulff-type boronate affinity chromatography, porous graphitic carbon chromatography and high-resolution mass spectrometry for the extraction, separation and indetification of cis-diol containing biomoleculesAs many cis-diol molecules of biological importance are present in low abundance in the samples while interfering substances are usually present in high abundance, specific capture and effective enrichment of target cis-diol biomolecules becomes a key and challenging step in the-omics analysis.Boronate affinity chromatography (BAC) is an important means for the specific capture of cis-diol biomolecules. We developped a Wulff-type boronate affinity monolithic capillary column for the on-line coupling with liquid chromatography-mass spectrometry (LC-MS) for the extraction, separation and identification of cis-diol containing biomolecules. A Wulff-type boronate affinity monolithic capillary column was used as solid phase extraction column for the selective trapping of nucleosides while a porous graphitic carbon (PGC) column is used as a secondary column for the separation. This liquid chromatography system is on-line coupled with nanospray ionization LTQ-Orbitrap XL MS for the analysis of urinary nucleosides, which could provide the accurate molecular weight of the analytes. Except for quantitative analysis of targeted compounds, LTQ-Orbitrap XL MS could provide non-targeted screening and accurate mass confirmation of unknow compounds. As the boronate affinity monolithic column can specifically trap the target molecules under neutral pH, urine sample can be directed loaded to the system. We obtained20kinds of nucleosides for human urine successfully. Since all of the extracted compounds contain cis-diol group, the on-line coupling methods can simplized the identification greatly.