Study On Phosphorylation And Glycosylation Of Proteins Based On Functional Hybrid Nanomaterials

As one of the most important and ubiquitous post-translational modifications (PTMs), protein phosphorylation plays a central role in eukaryotic cells involved in regulation of cell growth, division, migration, differentiation, and signaling. Therefore, study on protein phosphorylation may help to understand the physiological functions and the mechanism of protein. Up to date, the basic method to study protein phosphorylation is usually by analysis of peptides released from the protein through proteolysis. Mass spectrometry (MS) is a powerful technique for determining the phosphorylation profiles of proteins in phosphoproteome research because of its high sensitivity, high-throughput, and simplicity in identification of phosphorylation sites and quantification of changes in phosphorylation states. However, phosphopeptides can be suppressed by non-phosphopeptides in MS detection due to their low abundance and low ionization efficiency. The selective enrichment of phosphoproteins or phosphopeptides from complex mixtures, especially biosamples, is therefore essential for MS-based phosphoproteomics. Protein glycosylation is one of the most ubiquitous forms of post-translational modifications (PTM), it plays a crucial role in various biological processes, including cell growth, signal transduction, immune response and receptor activation. Glycoproteins have been used as therapeutic targets and biomarkers for cancer prognosis, diagnosis, and monitoring. Mass spectrometry (MS) has become a powerful tool for the analysis of glycopeptides or glycoproteins. However, the inherent low abundance of glycopeptides, the micro-heterogeneity of each glycosylation site and the ion suppression effect caused by the co-existence of nonglycopeptides make the detection of glycopeptides extremely difficult. Therefore, the selective enrichment of glycopeptides becomes imperative.In this study, we focused on preparing several different kinds of novel functionalized nanomaterials with large surface area and using them for selective enrichment of phosphopeptides and glycopeptides. This dissertation is divided into five parts.In Chapter 1, we frist summarized the background of protein phosphorylation and protein glycosylation in details. First, the physiological functions and basic research methods of protein phosphorylation were introduced. This review gives an overview over the most frequently used methods in isolation and detection of phosphopeptides and glycopeptides. Mass spectrometry is the most powerful method in study of protein phosphorylation and protein glycosylation. At last, the application of the functionalized nanomaterials in proteomics has been summarized.In Chapter 2, we prepared several IMAC nanomaterials by the simple dopamine chemistry for selective enrichment of phosphopeptides. Dopamine and its derived compounds, a mimic of 3,4-dihydroxy-L-phenylalanine (DOPA) found in the adhesive protein Mefp-5 (Mytilus edulis foot protein 5) secreted from mussels, has been found to be able to polymerize into a unique hydrophilic polydopamine (PD) that can adhere to a variety of substrates. This unique natural adhesive have attracted wide-spread interest due to its versatility for surface modification. The polydopamine coating exhibits an excellent environmental stability, good biocompatibility and water dispersibility. The novel IMAC materials were demonstrated to have high selectivity and sensitivity for the enrichment of phosphopeptides in complex samples such as protein digest and human serum sample. This work was expected to open up the promising strategy for a more efficient and sensitive tool for selective enrichment of phosphopeptides in phosphoproteome research.In Chapter 3, we synthesized several MOAC nanomaterials for selective enrichment of phosphopeptides. We used them to isolation and enrichment of the phosphopeptides from tryptic digestion of both standard proteins and real samples. The as-prepared MOAC nanomaterials have been demonstrated as a powerful tool for phosphoproteomics research.In Chapter 4, hierarchically ordered macro/mesoporous alumina (HOMMA) has been successfully synthesized as a nanoreactor with multi-functions, which combine in situ digestion and in situ enrichment via one simple integrated step but with high efficiency. By directly adding HOMMA into the conventional digestion system, enzyme can be absorbed into the nanopores quickly to greatly accelerete the digestion. At the same time, because of the chemo-affinity between phosphor groups and alumina, the specific phosphopeptides can be enriched in the nanopores. The HOMMA nanoreactor has performed to be effective for not only standard phosphoproteins but also complex samples. It could be expected that by rational design of macro/mesoporous materials, versatile nanoreactors with enhanced multiple biological functions and desired performance could be achieved, which may play important roles in enzymatic catalysis and modern proteomics.In Chapter 5, polyboronic-functionalized hierarchically ordered macro/mesoporous silica (HOMMS@PolyVPBA) was synthesized and applied to enrich phosphopeptides. Hierarchically ordered macro/mesoporous materials, with intriguing macropores structure, are especially desirable as the macroporous channels facilitate mass transport while mesopores provide high surface area and large pore volume. Thus, hierarchically ordered macro/mesoporous materials can provide excellent performance of mass transport and offer easier accessibility for guest object to framework binding sites, which can find substantial applications in catalysis, adsorption, separation and biotechnologies. HOMMS@PolyVPBA was applied to enrich glycopeptides with an ultrafast enrichment speed (within only 10 min), high specificity (a molar ratio as low as 1:50 of glycopeptides/non-glycopeptides could be selectively analysed) and the enrichment selectivity for glycopeptides is high, even for HRP digests at concentrations as low as 1 ng μL-1.In summary, the main contribution of this dissertation is that we initially synthesized several novel MAC and MOAC nanomaterials and successfully applied them in phosphopeptides and glycopeptides enrichment.