New Technology And New Method For Highly Selective High Sensitivity Glycopeptide Enrichment And Mass Spectrometry

This dissertation is focusing on the hot and tough issues of glycopeptide detection by mass spectrometry (MS) in glycoproteome and glycopeptidome research, such as the low abundance of glycopeptides, the interference of non-glycopeptides, and the low detection sensitivity. We have developed the boronic acid-functionalized mesoporous nanomaterials (MCM-41-GLYMO-APB, MCM-41-APTES-CPB) and the hydrazide-functionalized polymer magnetic nanocomposites (Fe3O4@PMAH) to achieve the highly selective enrichment and highly sensitive MS detection of glycopeptides. Finally, the glycopeptidomes of complex biological samples like rat serum and snake venom were highly specifically revealed by the boronic acid-functionalized mesoporous nanomaterials, as well as the glycoproteins from the colorectal cancer patient serum were identified by the hydrazide-functionalized polymer magnetic nanocomposites.As one of the most common, important and complex post-translational modifications (PTMs), the glycosylation of proteins is of an extremely important biological significance. Glycosylation can affect the three-dimensional structures of proteins or decide the transfer directions of proteins within the cells, and it plays an important role in the recognition between cells and matrixes, signal transduction, and the process of development and differentiation. To date, more than half of the discovered cancer biomarkers are glycosylated proteins or peptides; the carbohydrate changes are closely related to the initiation and progression of tumors. Therefore, the specific profiling of glycopeptides and glycoproteins are highly inclined toward the discovery of disease biomarkers and clinical diagnosis. However, despite more than50%proteins of the mammalian occurred glycosylation, glycopeptides usually exist at relatively low abundances (2%to5%) compared with non-glycosylated peptides. The glycan microheterogeneity further reduces the relative amount of glycopeptides and decreases the detection sensitivity. In the MS analysis, the signals of glycopeptides are susceptible to the existance of non-glycopeptides, therefore the study of glycosylation is challenging. How to highly selectively enrich glycopeptides from complex biological samples and make glycopeptides be detected by MS is a critical problem need to be firstly solved in glycoproteome research.Peptidome, the low-molecular-weight (LMW) subset of the proteome, has attracted increasing attention in recent years since it was first proposed in2001. A serum sample is believed to contain these LMW circulating proteins and peptides, which can be rich sources of information. The analysis of peptidome in biological samples via MS potentially provides diagnostic and prognostic information on cancer and other diseases. Snake venom is a complex mixture secreted from the venom gland containing many functional enzymes and peptides which have pharmacological activities. Venom can paralyze and kill the prey, but it is also a valuable resource for discovering new drugs. The venom peptidome research is very important, which is conducive to mining bioactive molecules for potentially therapeutic applications. However, only few reports on post-translational modifications of peptidome can be found although the awareness on the importance of the peptidome has greatly increased. Despite the potential of glycopeptidomics, some fundamental and serious barriers exist, hindering glycopeptidome research. First, the high dynamic range of proteomes in complex biological samples renders the analysis of glycopeptidome a very challenging task. Second, the molecular weight (MW) of the glycopeptidome is mostly lower than20kDa, which is similar to the peptidome, is difficult to be covered via traditional sample preparation techniques in proteomics. Lastly, glycopeptides usually exist at relatively low abundances (2%to5%) compared with non-glycosylated peptides. The glycan microheterogeneity further reduces the relative amount of glycopeptides and decreases the detection sensitivity. Therefore, a specific method to overcome the limitations of the traditional methods for glycopeptidome research needs to be developed.In this thesis, we will introduce our researches in five chapters.In the first chapter, we briefly reviewed the proceeding and challenges of peptidome, technologies in glycoproteomics, and the application of nanomaterials in glycoproteomics. The development and challenges of sample preparation methods in peptidomics, the comparison of separation and enrichment technologies in glycoproteomics, the development of a variety of nanomaterials applicable to glycoproteome enrichment were discussed in details. All of the background information provides theoretical and applicable support on doing researches.In the second chapter, the boronic acid-functionalized mesoporous nanomaterials (MCM-41-GLYMO-APB, MCM-41-APTES-CPB) were designed and synthesized to selectively enrich glycopeptides. The mesoporous materials MCM-41with a large specific surface area (≈1000m2/g) is suitable for the functionalization of boronic acid groups, the pore size (2.1～2.7nm) and hexagonal columnar pore structure which has the size exclusion effect make it ideal for peptidome research, therefore the boronic acid-functionalized MCM-41may be suitable for glycopeptidome research in biological samples. In the first part, a boronic acid-functionalized mesoporous nanomaterial (MCM-41-GLYMO-APB) was prepared in aqueous solution and characterized by FT-IR. The conditions of enriching glycopeptides in mixed digests of standard proteins by MCM-41-GLYMO-APB were compared and optimized. In the second part, a modified boronic acid-functionalized mesoporous nanomaterials (MCM-41-APTES-CPB) was synthesized in the organic phase. The MCM-41-APTES-CPB exhibited excellent selectivity by analyzing glycopeptides in the mixture of glycopeptides/non-glycopeptides at molar ratio of1:100, extreme sensitivity (the limit of detection was at the fmol level), good binding capacity (40mg·g-1), as well as the high post-enrichment recovery of glycopeptides (up to88.10%). Meanwhile, the highly ordered hexagonal cylindrical mesoporous structure of MCM-41-APTES-CPB provided the possibility of being used for the glycopeptidome research in biological samples.In the third chapter, we established a method revealed the rat serum glycopeptidome by the boronic acid-functionalized mesoporous nanomaterials to overcome the deficiencies of the current research methods in serum glycopeptidomics. Mesoporous materials with glycopeptide-suitable pore size and rich boronic acid groups would be the ideal materials for glycopeptidome enrichment. The attractive features of the prepared MCM-41-APTES-CPB well met the requirements of serum glycopeptidome enrichment. Firstly, it has a small pore entrance size along with uniform mesopores so that high molecular weight (HMW) proteins are excluded from entering the pores. The size-exclusion ability was shown to exclude proteins with molecular weights (MWs) larger than12kDa in standard protein mixtures. Secondly, abundant boronic acid groups were grafted on its internal surface; therefore, once the peptides and the low molecular weight (LMW) proteins enter the pores, the glycopeptides and the LMW glycoproteins can be selectively captured by the boronic acid groups. Thirdly, the combination of glycopeptide-suitable pore entrance size and glyco-selective effects of boronic acid groups possesses the size-exclusion ability to selectively capture the glycopeptides and the LMW glycoproteins from complex biological samples. Finally, a novel method for glycopeptidome research combined with glycopeptide enrichment by the boronic acid-functionalized mesoporous silica and MS analysis was established. Rat serum glycopeptidome was revealed with15 unique glycosylation sites mapped to15different endogenous glycopeptides identified.In the fourth chapter, there has been few study on snake venom glycopeptidome, therefore we established a method revealed the snake venom glycopeptidome by the boronic acid-functionalized mesoporous nanomaterials. In the case of venom glycopeptides, such studies are important because of the potential to discover bioactive molecules with therapeutical application. Although studies on snake venom proteomes are growing rapidly, the characterization of venom peptidomes do not progress as fast, and the snake venom glycopeptidome even has been seldomly studied yet. We established a method revealed the snake venom glycopeptidome by boronic acid-functionalized mesoporous nanomaterials. In order to verify the effectiveness of this method, we also employed other four different methods to reveal the venom glycopeptidome. These four different methods combined the technologies in peptidome extraction and glycopeptide enrichment, including acetonitrile precipitation, ultrafiltration, acetonitrile precipitation combined with hydrazide resin enrichment, and ultrafiltration combined with hydrazide resin enrichment. The results showed that the revelation of venom glycopeptidome by MCM-41-APTES-CPB was the best among these five methods. The largest number of identified glycopeptides and the highest specificity was obtained with this established method. Snake venom glycopeptidome was revealed with15unique glycosylation sites mapped to15different endogenous glycopeptides identified via these methods.In the fifth chapter, a hydrazide-functionalized polymer magnetic nanocomposites was prepared and applied in the identification of glycoproteins from the colorectal cancer patient serum. This prepared material combined the advantages of magnetic core and high density of hydrazide groups which makes it has the potential to apply in the large-scale, high throughput and automated sample processing. Firstly, the abundant hydrazide groups on the surface of this material make it enrich glycopeptides with high specificity. Secondly, the magnetic core of this material makes it being easily separated. The preparation process was as follows: polymer-coated magnetic nanocomposites with a large number of carboxyl end was prepared by distillation precipitation polymerization, then adipic acid dihydrazide was grafted to the carboxy on the surface of the polymer-coated magnetic nanocomposites to give a hydrazide-functionalized polymer magnetic nanocomposites (Fe3O4@PMAH). The density of hydrazide groups was154±11μmol/g, and the magnetic saturation value was25emu/g. Compared to hydrazine resin, Fe3O4@PMAH improved more than five times the signal-to-noise ratio of enriched glycopeptides. Finally, Fe3O4@PMAH was applied to the enrichment of glycopeptides from the digest of colorectal cancer patient serum. There was a good reproducibility between three repeated experiments, totally175unique glycopeptides and181glycosylation sites corresponding to63unique glycoproteins were identified. The specificities of the enriched glycopeptides and glycoproteins were70%and80%, respectively.