A New Method For Identification Of Sucrose Peptide Based On Functionalized Magnetic Nanomaterials And Mass Spectrometry

As one of the most ubiquitous and most complicated post-translation modifications of proteins, glycosylation has been estimated to be involved in more than half of cancer biomarkers. Protein glycosylation is essential for a wealth of biological events including protein folding, intracellular sorting, secretion, uptake, as well as cell and host-microbial recognition. However, due to the low abundance as well as the low ionization efficiency of glycopeptides, the profiling of glycosylation has often been restrained by the signal suppression of coexisting non-glycosylated peptides in mass spectra. Besides, the microheterogeneity of glycosylation is of enormous complexity, which further decreases the proportion of glycopeptides among its digests. As a consequence, the pre-fractionation of glycoproteins/glycopeptides from complex biological samples with high specificity and high sensitivity is of primary importance.Currently, continuous efforts have been devoted to develop strategies for isolation of glycopetides/glycoproteins from complex biological samples, while any of them are far from perfect. In this dissertation, we aimed to develop novel protocols for the separation of glycopeptides which could address the above mentioned challenges. In the first chapter, the introduction of glycosylation had been presented, which was followed by a brief summary of the currently used methods for the enrichment of glycopeptides/glycoproteins. 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 functionalized nanomaterials for prefraction of glycopeptides and glcyproteins.Chapter2demonstrated an accessible protocol for the selective extraction of N-linked glycopeptides. Due to the unsurpassable specificity of solid phase extraction by hydrazide chemistry, it had drawn extensive attention. However, it turned out to be sample-consuming and expensive, which inevitably became the bottleneck of such strategy. Thus we proposed to develop a novel separation technique based on the conjugation of aldehydes from oxidized glycopeptides and amine-group on the magnetic nanoparticles via reductive amination reaction. Compared with the traditional solid-phase extraction methods realized by hydrzide chemistry, a desalting step was no longer needed in such protocol, which facilitated the realization of higher sensitivity. In addition, such strategy could render excellent enrichment performance on the premise of reducing coupling time by more than double. Thus the advantages of such strategy are obvious:on the one hand, the synthetic procedure of materials is easily accessible and cost-effective. On the other hand, the established isolation protocol was compatible for MS analysis and possessed the virtues of high sensitivity and high specificity. In this research, we especially focused on the synthesis of amine-functionalized nanoparticles, and systematically investigated the influence of modified functional groups and matrix materials on the isolation performance so as to find the optimized separation conditions. Then a brand-new protocol based on amine-functionalized magnetic nanoparticles for glycoprotein/glycopeptides enrichment combined with bio-MS technologies could be established and applied for the characterization of glycoproteome of human colorectal cancer serum and mouse liver tissue.In chapter3, aminooxy end-functionalized magnetic nanoparticles had been successfully synthesized for the isolation of glycosylated peptides. In this strategy, glycoproteins were digested and oxidized in the same way as the method used in hydrazide chemistry. After incubation, glycopeptides could be immobilized on the aminooxy end-functionalized magnetic nanoparticles through the formation of stable oxime bond and non-glycosylated peptides could be washed away. Specific enrichment performance could be achieved when the digests of myoglobin were mixed with the asialofetuin digests at the mole ratio of10:1. Finally, the established protocol was applied for the profiling of N-linked glycosylation of rat serum. This protocol not only eliminated the desalting step after the oxidation of glycopeptides, but also omitted the usage of reducing reagent, thus largely contributed to the ease of operation.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 glycosylated peptides and proteins. Two brand-new solid phase extraction methods based on functional nanoparticles have been established and introduced into the field of glycoproteome research. Both of them inherits the virtue of easily accessible and could achieve the separation with high sensitivity and high specificity. As a consequence, these protocols are believed to be promising for the mapping of N-linked glycosylation of complex biological samples and shed new light upon glycosylation characterization.