| Based on proteome research background and development trend of nanomaterials, the research interest of this work focused on combining various techniques, including nanotechnology, chemical derivatization, signal amplification etc., with traditional bioanalytical methods in proteome research, to develop a series of novel techniques and methods to resolve existing problems in specific enrichment and mass spectrometric detection of phosphorylated proteins and glycosylated proteins in post-translational proteome research. This dissertation is divided into five parts.Chapter 1 summarized current situation and existing problems of pretreatment technologies prior to mass spectrometric analysis in proteomics, key advances in the development of low-abundance protein enrichment and preconcentration techniques. Since phosphorylation and glycosylation are both highly important post-translational modifications and key challenge in phosphorproteome and glycoproteome research is the development of fast and effective enrichment strategies for high-throughput identification, we highlighted several examples on various types of enrichment methods that have been utilized to specifically capture these modified proteins for subsequent mass spectrometric analysis. In the past few years, the introduction of nanoparticles into proteome research has accelerated the development of enrichment methods, so we also summarized recent developments of using different functional nanomaterials for pre-concentration of low-abundance peptides/proteins, including those containing post-translational modifications, such as phosphorylation and glycosylation, prior to mass spectrometric analysis in details. The intention and meaning of this dissertation were explained in this chapter.In chapter 2, a novel strategy based on carboxy group derivatization is presented for specific characterization of phosphopeptides. Electron-transfer dissociation (ETD), a relatively new dissociation method that was developed recently, has the attractive feature of independence of amide bond protonation and shows the advantage in preserving the information about post-translational modifications during peptide fragmentation, thus providing an efficient way for sequencing phosphorylated peptides/proteins. However, since ETD only works with peptides having more than one positive charge, one of the most challenging tasks posed in phosphorylation analysis is to increase ion charge state. By tagging the carboxy group with 1-(2-pyrimidyl) piperazine (PP), the ion charge states of phosphopeptides can be largely enhanced, showing great advantages for sequencing phosphorylated peptides with electron-transfer dissociation MS. For peptides that mainly hold one positive charge from electrospray ion source, they are not suited for ETD-MS/MS analysis. However, after PP-derivatization, the primary species became those who hold double charges. Thus, it was not a barrier anymore for them to be applied to ETD-MS/MS analysis. Besides, by blocking the carboxy group, the PP-derivatization method can also be employed to eliminate non-specific interactions between acidic residues and TiO2, which is the major competitive process during the TiO2-based phosphorylated peptides/proteins enrichment strategy, thus greatly increasing the selectivity toward phosphopeptides/phosphoproteins. Moreover, being tagged with a hydrophobic group, the retention time of phosphopeptides in RPLC can be prolonged, overcoming the difficulty of separating phosphopeptides in RPLC-based approach. Together with several other advantages, such as ease of handling, rapid reaction time, broad applicability and good reproducibility, this PP-derivatization method is promising for high-throughput phosphoproteome research.In chapter 3, a core-satellite-structured composite material has been successfully synthesized for capturing glycosylated peptides or proteins. The inherent low abundance of glycoproteins and the microheterogeneity of each glycosylation site make the enrichment procedure before MS analysis a prerequisite. Although a variety of methods are available for glycopeptides or glycoprotein enrichment, the complete mapping of glycoproteome is still a challenging task. Nanoparticles are attracting considerable interest on account of their significant potential in biotechnology and biomedicine for diagnostic and therapeutic applications. Recently, particular attention has been paid to the synthesis of composite nanoparticles that have both the biocompatibility and surface chemistry of gold and the magnetic properties of superparamagnetic particles. The novel hybrid material synthesized in our study is composed of a silicacoated ferrite "core" and numerous "satellites" of gold nanoparticles with lots of "anchors". The anchor,3-aminophenylboronic acid, designed for capturing target molecules, is highly specific toward glycosylated species. The long organic chains bridging the gold surface and the anchors could reduce the steric hindrance among the bound molecules and suppress nonspecific bindings. Due to the excellent structure of the current material, the trap-and-release enrichment of glycosylated samples is quite simple, specific, and effective. Indeed, the composite nanoparticles could be used for enriching glycosylated peptides and proteins with very low concentrations, and the enriched samples can be easily separated from bulk solution by a magnet. By using this strategy, the recovery of glycopeptides and glycoproteins after enrichment were found to be 85.9 and 71.6% separately, whereas the adsorption capacity of the composite nanoparticles was proven to be more than 79 mg of glycoproteins per gram of the material. Moreover, the new composite nanoparticles were applied to enrich glycosylated proteins from human colorectal cancer tissues for identification of N-glycosylation sites. In all,194 unique glycosylation sites mapped to 155 different glycoproteins have been identified, of which 165 sites (85.1%) were newly identified.Chapter 4 presents a highly sensitive glycoprotein detection method, by using magnetic microparticle to isolate glycoproteins and gold nanoparticle (AuNP) to amplify the signal intensity with mass spectrometer. The homemade AuNP is a perfect signal reporter since it gives intensive peak of Au2+ and negligible background noise with mass spectrometer. Additionally, each AuNP contains more than 64000 gold atoms so it is potentially capable of amplifying the signal intensity for several orders of magnitude once adopting Au2+ as the signal tag instead of detecting intact glycoproteins. To fulfill this purpose, carboxy group functionalized AuNP was graft onto amino groups of glycoproteins after isolating glycoproteins with boronic acid functionalized magnetic microparticles. The AuNPs bound glycoproteins were then eluted and applied to mass spectrometer. Using this strategy, the limit of detection (LOD) of horseradish peroxidase was improved to 7.695 fM (S/N=3) while that for another two glycoproteins (asialofetuin and ribonuclease b) were 18.87 fM (S/N=3) and 322.2 fM (S/N=3) respectively. Moreover, this method shows good selectivity encountering glycans and non-glycoproteins.In chapter 5, amine modified magnetic nanoparticles were synthesized and applied to selectively enrich glycosylated peptides from tryptic glycoprotein digestion. In this strategy, glycoproteins are digested into peptides firstly, containing both glycosylated peptides and nonglycosylated peptides. Then, the cis-diol groups of carbohydrates in glycopeptides are oxidized into aldehydes, which can form covalent bonds with amine groups immobilized on magnetic nanoparticles. Non-glycosylated peptides are washed away, whereas the glycosylated peptides still remain on the matrix material. PNGase F is then used to release the N-glycopeptides and the resulting peptides are subjected to mass spectrometric analysis. The washing buffer and washing condition are carefully optimized in this study. Finally, we realized selective enrichment of glycopeptides from tryptic digests of standard glycoproteins.In summary, the main contribution of this dissertation is to develop and provide several effective techniques and methods to resolve the difficulties in specific enrichment and mass spectrometric detection of phosphorylated proteins and glycosylated proteins. Several functional nanomaterials have been synthesized for different research requirements, and novel technologies have been introduced into proteome research fields. We aim at exploring and finding out new techniques in the pre-concentration and MS analysis of phosphorylated and glycosylated proteins, so that more breakthroughs can be obtained in post-translational proteome research. |
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