Study On New Technology And New Method Of Sugar Proteomics / Sugar Histology Based On Mass Spectrometry

The contents of this dissertation relates to an interdisciplinary research involved in analytical chemistry, chemical biology and so on. On the basis of bio-MS, the existing methods related to glycoproteomics/glycomics were compared, improved and innovated. Moreover, several novel methods and technologies were developed for glycoprotein enrichment, mass spectrometry identification and labeling quantification, and were also applied in analysis of protein glycosylation of complex biological samples. The developed methods and technologies can be used for qualitative and quantitative analyses of protein glycosylation modification in important biological samples accurately and efficiently, including analysis of glycoprotein/glycopeptide, glycosylation site, glycan, and so on.With the development of proteomics, protein post-translational modifications have been paid more and more attention by scientists. As one of the most common and important post-translational modifications, glycosylation research has become a hot field in protein post-translational modification research. Base on the bio-MS and proteomics, the protein glycosylation study mainly contains two parts, glycoproteomics and glycomics. In recent years, with the development of research methods in glycoproteomics and glycomics, an increasing number of protein glycosylation information was obtained. However, because of the complexity and variability of protein glycosylation, as well as the limitations of existing methods and technologies, much is still unknown about the protein glycosylation. For example, more than 50% of all proteins have been estimated to be glycosylated, but only 10% of them have been annotated as glycoproteins in in the protein database and most potential glycoproteins have not yet been discovered. Another example is that some glycoproteins and glycans are known to be changed in abundance in many pathological conditions, however, many things including how the glycosylation changed, why they changed, and the relative/absolute content of glycoproteins/glycans are still unclear. Therefore, glycosylation research faces challenges and opportunities, and bio-MS-based glycosylation methods and technologies still need further advancing and progress.In connection with the qualitation and quantitation of glycoprotein and glycan and on the basis on bio-MS, this dissertation utilized, analyzed and compared the traditional glycoproteomics/glycomics methods and strategies, and creatively developed several novel and effective glycoprotein enrichment, mass spectrometry identification and labeling quantification methods and technologies, and successfully applied them to complex biological samples, which can partly solve the existent technical problems in glycoproteomics and glycomics, and provide analytical strategies for this field. The major contributions of this dissertation are as follows:(1) The traditional glycoproteomics and glycomics methods were for the first time used in the high throughput exploration of N-glycosylation in three most abundant snake venoms in Asia. The N-glycoproteins and N-glycans form the three snake venoms were comprehensively identified and analyzed through optimization and combination of N-glycoprotein and N-glycan identification methods. Moreover, the largest set N-glycosylation data of snake venom to date was established.(2) Two conventional enrichment methods, lectin and TiO2 were compared for the first time. Furthermore, a new strategy, peptide immobilized pH gradient isoelectric focusing (IPG-IEF) assistant TiO2 chromatography (PIAT), was developed for the highly efficient enrichment of sialoglycopeptides. The largest number of sialoglycoproteins were identified with the new developed method, which solve the problem of difficulties in glycoprotein enrichment and identification, and prove a novel method for the sialoglycprotein enrichment.(3) Sodium borohydride assistant PNGase F-catalyzed N-glycan dual labeling quantitation method was creatively developed. This method not only improved the stability of glycan 16O/18O labeling, but also increased the mass gap between 16O/18O labeled glycan to 3 Da, and thus largely reduced interference of isotope overlaps, filling a technical blank in PNGase F-catalyzed N-glycan labeling quantitation technologies. Moreover, this method was used for N-glycan quantitation in hepatocellular carcinoma (HCC) associated human serum, and some significant changed N-glycans were identified, which laid the foundation for further potential diagnostic biomarkers exploration and N-glycan function studies.(4) By combination of β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) enrichment method, mass spectrometry and immunological methods, for the first time, some mitochondrial proteins in rat liver were definitely not only identified but also validated to be O-GlcNAcylated. In addition, "cross talk" relationship between O-GlcNAcylation and phosphorylation was implied to occur in mitochondria.Firstly this dissertation summarized the protein glycosylation, described the progress of technology in glycosylation study, presented the actualities and challenges from this field, and point out the aims and significance of this dissertation. Then the dissertation narrated in the following four parts:The high throughput identification of snake venom glycosylation with the combination of glycoprtoteomics and glycomics strategiesSnake venom is a complex cocktail including a variety of biological active proteins and proteinaceous components, which have considerable medical and pharmacological importance. N-glycosylation is widely implicated as a common modification on numerous venom proteins and impacts the in vivo venomic functions. However, systematic N-glycome and N-glycoproteome survey on snake venoms have not been undertaken.In this part, employing combination of N-glycomics and N-glycoproteomics strategies, we for the first time explored the N-glycosylation including both N-glycoproteins and N-glycans in three venoms, Agkistrodon blomhoffii, Naja naja atra Cantor and Vipera russelii siamensis Smith which are amongst the most abundant venomous snakes in Asia. A total of 9,8 and 18 N-glycoproteins,115,100 and 95 N-glycans were respectively identified in Agkistrodon blomhoffii, Naja naja atra Cantor and Vipera russelii siamensis Smith venoms. Most of these N-glycoproteins and N-glycans were discovered for the first time, providing much novel glycosylation information for snake protein database. In addition, we found that the overlaps of N-glycoproteins and N-glycans among the three venoms were small, indicating great differences existed in different snake venoms. This investigation has the following contributions:(1) The investigation in this part not only provided us a large number of N-glycoproteins and N-glycans in the three snake venoms, but also provided us with basis for the comprehensive understanding of venoms variation. (2) Through the analysis, we found that complex and abundant N-glycans increased the complexity and variety of the three venoms, and glycosylation level seemed to be distinct among the snakes, which is the major reason of venom differences. (3) The utilization of N-glycoproteomics and N-glycomics methods in this investigation laid a solid experimental foundation for the follow-up new methods development in this dissertation.peptide IPG-IEF assistant TiO2 chromatography method for Enhanced sialoglycopeptide enrichment and large-scale sialoglycoprotein identificationBeing an important role in a broad range of biological and pathological processes, for example, the over-representation of sialic acids on the surface of glycoproteins is thought to be associated with cancer, sialylation has been drawing in wide interest. The efficient sialoglycopeptides enrichment methods are therefore attracted considerable attention. Many sialoglycoproteins/sialoglycoepeptides isolation and enrichment methods have been developed. However, because of the complexity and heterogeneity of glycosylation, none of these methods can capture all the sialoglycoproteins/sialoglycopeptides from complex biological mixtures. Therefore, more efficient methods of isolating sialoglycopeptides from mixture are arousing attention.In this part, we for first time compared two conventional enrichment methods, lectin and TiO2, and analyzed their characteristics. Furthermore, considering the highly negatively charged nature of sialic acids, we developed a new strategy, peptide immobilized pH gradient isoelectric focusing (IPG-IEF) assistant TiO2 chromatography (PIAT), for the highly efficient enrichment of sialoglycopeptides. In this method, peptides were first separated into 24 fractions using peptide IPG-IEF. Sialoglycopeptides were relatively concentrated in low-pH fractions of the immobilized pH strips, then the resulting fractions were further enriched with TiO2 chromatography and followed by LC-ESI-MS/MS analysis. As a result,614 N-glycosylation sites were identified in 582 sialoglycopeptides within 322 sialoglycoproteins from rat liver using PIAT. To our knowledge, this work represents one of the most comprehensive sialoglycoproteomic analysis in general and exhibits the largest database of sialoglycoproteome in rat liver currently. Compared with traditional enrichment methods, PIAT exhibits better enrichment capacity of sialoglycopeptides with different pI, molecular weight and subcellular distribution. The new strategy introduced in this part exhibits high efficiency and universality in the sialoglycopeptides enrichment, and is a powerful tool for sialoglycoteome exploration. This investigation has the following contributions:(1) Two conventional enrichment methods, lectin and TiO2, were compared for the first time, which provide effective references for different experimental requirements; (2) A new sialoglycopeptide enrichment strategy was developed and applied in large-scale of sialoglycoproteins identification, which provides an alternative powerful tool for sialoglycoteome exploration. (3) Compared with traditional enrichment methods, PIAT shows higher efficiency and universality; (4) This work represents the largest database of sialoglycoproteome in rat liver currently. The results provide experimental evidence for numerous potential N-glycosylation sites as well as a number of new N-glycosylation sites for the Swiss-Prot database.N-glycan dual labeling method for credible N-glycan quantitationGlycosylation quantitation is also an important field in glycosylation research. Quantitative glycomics has been developed along with the advancing of qualitative glycomics and glycobiology, aiming at studying N-glycan changes in specific physiological and pathological process, which is important for exploring potential biomarkers in diseases. Traditional glycomic quantitation mainly rely on comparing the chromatography peak area of derivative glycans. However, with the development of mass spectrometry (MS) techniques, glycan quantitation based on MS has provided new perspectives. Isotopic labeling is considered to be an important method in relative glycan quantitation. Recently, many isotopic labeling glycan quantitation methods have been developed, including reducing end isotopic labeling, permethylation isotopic labeling and metabolism labeling method. Previously our lab for the first time realized the enzyme-catalyzed 18O N-glycan labeling quantitation method, which is an important technology in quantitative glycomics. However, this kind of quantitative method has some problems, for example, the labeled 18O is not stable and the overlap of isotopes may interfere quantitative results.In this part, we for the first time treat the N-glycans with NaBH4 after the PNGase F-catalyzed N-glycan 18O labeling. We found that the N-glycan 0 labeling become more stable having no exchange with 16O in normal H2O after the N-glycan end reduced with NaBH4. In addition, we creatively developed sodium borohydride assistant PNGase F-catalyzed N-glycan dual labeling method for N-glycan quantitation by treating the PNGase F-catalyzed 16O/18O-labeled N-glycans with NaBH4/NaBD4. This method expands the molecular difference to 3 Da between the labeled glycan pairs, which reduce the overlap of isotopic clusters. The novel method optimize the PNGase F-catalyzed N-glycan labeling, making glycan 18O-labeling more stable and glycan quantitation more accurate. We also applied this method for quantitation of N-glycans in in sera from healthy individuals and patients diagnosed with hepatocellular carcinoma (HCC), which indicated the efficiency and potential application value of this method. This investigation has the following contributions: (1) NaBH4 and NaBD4 were first introduce into N-glycan labeling, which make the N-glycan 16O/18O labeling become more stable; (2) NaBH4/NaBD4 assistant PNGase F-catalyzed N-glycan dual labeling method was creatively developed. This method can expand the molecular difference to 3 Da, which greatly reduce interference of isotopic clusters overlapping; (3) This N-glycan dual labeling method exhibited high labeling efficiency, quantitative accuracy and reproducibility, and can be applied in complex biological sample quantitation, undoubtedly providing a powerful method for quantitative glycomics; (4) Quantitative analysis of HCC associated human serum N-glycans was performed by this method. Several changes were found, which laid the foundation for further potential diagnostic biomarkers exploration.Discovery and confirmation of O-GlcNAcylated proteins in rat liver mitochondria by combination of mass Spectrometry and immunological MethodsO-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. O-GlcNAcylation involves in wide biological processes, such as cell signaling, transcription, translation, protein degradation, cell cycle, and so on. Abnormal O-GlcNAc glycosylation is associated with many diseases, such as diabetes, Parkinson’s disease, Alzheimer’s disease, and so on. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid.In this part, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function. In addition, "cross talk" relationship between O-GlcNAcylation and phosphorylation was analyzed implied to occur in mitochondria, which is the an important biological question and worth studying in future. This investigation has the following contributions:(1) For the first time, in highly purified mitochondrial proteins of rat liver, we not only discovered but also confirmed a number of O-GlcNAcylated proteins; (2) The identification results provided a direct and unambiguous evidence for O-GlcNAcylation sites mapping in mitochondria; (3) The same as in nuclear and cytoplasm, "cross talk" relationship between O-GlcNAcylation and phosphorylation was implied to occur in mitochondria; (4) O-GlcNAcylated mitochondrial proteins play important roles in organism; (5) Although much remains unknown about the function of O-GlcNAcylation and the molecular mechanism of the interplay between O-GlcNAc and O-phosphate in mitochondria, the results in this part indicate a previously unrecognized and potentially significant role of O-GlcNAcylation in mitochondria and will arouse more detailed studies.