New Technologies And Methodologies Of Proteomics Based On Detonation Nanodiamond

Base on the proteome research background and the special property of nanodiamond (dNDs), the research interest of this work focus on the developing a series of techniques and approaches to resolve current problems in the pretreatment of low-abundance proteins/peptides, efficient digestion of proteins and the special enrichment of post-translational proteins/peptides. Firstly, the approach of concentration of low abundance peptides/proteins was developed by dNDs. It has shown good enrichment efficiency and celerity. Therefore, dNDs was applied for the research of the differential expression proteomic study of nephrogenesis in intrauterine growth restriction and the peptidome of HCC serum. Secondly, dNDs was applied as the MALDI support for increase the ionization efficiency of peptides, and further enhance the credibility of protein identification. Thirdly, due to the special activity functional group on the surface of dNDs, the immobilization of enzyme was successfully realized, and further enhanced the efficiency of protein proteolysis. And applied the property of dNDs, the functionalization of dNDs with amino boronic acid was realized and applied for the identification of glycopeptides in the fraction of the extract of rat liver. At last, dNDs was layer-by-layer functionalized on the magnetic polymer microspheres with the assistant of PL. And the facile synthesis and the convenient and efficient enrichment process of the novel Fe3O4@SiO2@[dNDs]n make it promising candidate for isolation of peptides/protein in complex biological samples, even the seretome of cell. This dissertation is divided into six parts.In chapter 1, advances in current protein separation and concentration techniques, the research and application of dNDs were summarized in details. The intention and meaning of this dissertation were explained.In chapter 2, detonation nanodiamond (dND) was firstly employed as adsorbent for pretreatment of peptides in dilute/contaminated sample solution. Detonation nanodiamond showed high efficiency for isolating and enriching peptides in a wide pH range. Remarkably, good tolerance capability toward salts and detergents could be achieved by using dNDs. Due to the inherent specificities of dNDs, dND-bound peptides could be directly analyzed by MALDI-TOF MS, so as to avoid the elution step and reduce sample loss. This pretreatment method also exhibited a better performance for protein identification compared to solvent evaporation and Ziptip pretreatment approach. Meanwhile, dNDs can be successfully applied to in the peptidome of HCC serum and the comparative proteomics study of nephrogenesis in intrauterine growth restriction (IUGR). In seru of HCC, fibinopeptide A was special highly expression, which offer referenced data for further research. At the same time, in the comparative proteomics research of IUGR, these 21 protein spots were preliminarily identified and were structural molecules, including vimentin, perlecan, gamma-actin and cytokeratin 10, transcription regulators, transporter proteins, enzymes, and so on. These proteins were involved primarily in energy metabolism, oxidation and reduction, signal transduction, cell proliferation and apoptosis. Data from this study may provide, at least partly, evidence that abnormality of metabolism, imbalance of redox and apoptosis, and disorder of cellular signal and cell proliferation may be the major mechanisms responsible for abnormal nephrogenesis in IUGR.In chapter 3, we report detonation nanodiamond (dND) (normal size 3-10 nm) support for MALDI-MS target, over which a cyano-4-hydrocinnamic acid (CCA) crystallizes evenly. Good reproducibility of relative peak intensity (R.S.D. less than 11.8%) among sample spot (from ring to center) is achieved on dND support. Therefore, the search for "hot spots" during the analysis is not necessary, which is supporting for the automatic acquisition of data. Due to high absorbability of energy from the laser, the dND support improves ionization efficiency of samples. In general, the sensitivity of MS obtained on dND support can be enhanced three to four times compared to the conventional MALDI sample preparation technique. Sensitivity obtained on dND support ranges from 62.5 amol of Arg-vasopressin standard peptide to 1.0 fmol of myoglobin tryptic peptide mixture. Reduced spot size and increased sensitivity in MALDI-MS are also accomplished by dND support. With the reduce of spot size, the signal intensity of cytochrome c (Cyt c) tryptic peptide obtained on dND support is at least seven times greater than it acquired on stainless steel. And dND support has been found better tolerance for salt (up to 500mM NaCl) to MALDI-MS analysis. All these properties make dND support a valuable tool for MALDI-MS identification of proteins.In chapter 4, immobilization of enzyme on detonation nanodiamond (dND,3-10 nm) and its application for efficient proteolysis have been demonstrated. By evaluation of the Michaelis constant (Km) and maximum velocity (Vmax) of immobilized enzyme, its activity was not impaired significantly by immobilization. And enzyme immobilized on dNDs exhibited much better thermal and chemical stabilities than its free counterpart and maintained high activity even after 10 times reuse. The efficient proteolysis by trypsin immobilized on dNDs (dND-trypsin) is demonstrated with the digestion of myoglobin (or other model protein) in a short time (5 min). Large numbers of identified peptides obtained by dNDs-trypsin enable a higher degree of sequence coverage and more positive identification of proteins than those obtained by in-solution digestion and the commercial immobilized trypsin beads, respectively. Moreover, immobilization of peptide-N-glycosidase F (PNGase F) on dNDs was realized and resulted in faster sequential glycosidase digestion of glycopeptides in less than 10 min.In chapter 5, we firstly present the functionalization of detonation nanodiamond (dND) with aminophenylboronic acid (APBA) for the specific and efficient capture of glycopeptides from complex sample. Glycopeptides specifically captured by dND-PL/PEG-APBA particles could be directly applied for analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Due to small particle size and abundance surface functional groups of dNDs, dNDs-PL/PEG-APBA particles provides better selective specificity and enriching efficiency for glycopeptides than that employed by the commercial boronic acid functionalized magnetic particles. In comparsion with direct and traditional analysis, the specific enriching approach enabled 2 orders of magnitude improvement in the detection limit of glycopeptides with 250amol/μL. In combination with LC-MS, the approach was applied to characterize one fraction of the digests of mouse liver extract, where 40 N-glycosylation peptides within unique 36 glycoproteins.In chapter 6, a novel magnetic silica nanoparticle functionalized with layer-by-layer detonation nanodiamonds (dNDs) were prepared by coating single submicron-size magnetite particle with silica and subsequent modification with detonation nanodiamond by the diimide-activated amidation of detonation nanodiamond-bound carboxylic acids assisted by poly-1-lysine. With the characterization of FT-IR and TEM analysis, the resulting layer-by-layer dNDs functionalized magnetic silica microspheres (Fe3O4@SiO2@[dNDs]n) exhibited well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersiblity and excellent magnetic responsibility. As a result of their excellent magnetic property, Fe3O4@SiO2@[dNDs]n particles were successfully applied for convenient, fast and efficient pretreatment of low-abundance peptides/proteins from high-diluted sample solution. The signal intensity could be improved by at least two to three orders of magnitude. Even in high salt concentration solution, peptides/proteins could also be isolated effectively. The experiment results were almost the same with the results gained using dNDs, but shown more effectively efficiency with the magnetic core. The facile synthesis and the convenient and efficient enrichment process of the novel Fe3O4@SiO2@[dNDs]n make it promising candidate for isolation of protein in secretome. Combination of the pretreatment approach of Fe3O4@SiO2@[dNDs]n and iTRAQ technology, the host response to HBV using an inducible HBV-producing cell line (HepAD38) was research by the comparative protein expression in the secretome of HepAD38 with and without tetracycline.In summary, the main contributes of this dissertation is to develop novel techniques and approach with assistant of dNDs and found some new pretreatment methods for low-abundance peptides/proteins and specific enrichment of glycosylated proteins/peptides. We aim at exploring and finding out new techniques in the separtion and concentration of proteome research fields, so that more breakthroughs can be obtained in the proteome research study.