Based On The Goal Of Biological Mass Spectrometry And Lectin Liquid Phase Chips Glycoprotein N - Sugar Dissolve The Analysis

This thesis presents an interdisciplinary research involved in analytical chemistry, chemical biology and biological chemistry. The application of mass spectrometry (MS), molecular biology technology and the innovation of lectin microarray have been achieved to efficiently reveal the glycosylation of target glycoprotein, including glycosylation sites, glycan structure and the analysis of glycosylation alteration.Glycosylation is one of the most important and universal protein post-translational modifications (PTMs) and plays an important role in differentiation, development, the metastasis of tumor, fertilization, immunity, infection and regeneration. The alteration of target glycoproteins provides crucial information for tumor early diagnosis, process monitoring and prognostic evaluation. Therefore, the glycosylation of target protein should be analyzed to reveal its important function. However, studies of the glycosylation of target glycoproteins have often been hampered by low abundance. Several glycoproteins have been expressed with a tag in human cell lines to obtain sufficient abundance for further assay. Several complementary approaches such as mass spectrometry, site-directed mutagenesis, permethylation and lectin blots were used on both glycosylation site and glycan structure analyses to provide a complete understanding of target protein glycosylation. However, it does not supply enough information to focus on the glycan structure. Various kinds of microarray such as antibody microarray, lectin microarray and glycan microarray have been developed for clinical diagnosis of glyco-biomarkers. For example, lectin microarray has been crucial for the disease diagnosis because of its high-throughput and high-sensitivity. However, the sensitivity, reproducibility, and throughput of lectin microarray should still be constantly improved to meet the increasing demand of clinical applications. The application of novel array system is still needed. The last but not the least, the separation, detection and annotation of oligosaccharides should be improved for the comprehensive analysis of glycan structure.The major contributions of this work are as follows:(1) The N-glycosylation pattern of recombinant human CD82(KAI1) was revealed for the first time. CD82was expressed in HEK-293T cells with a flag tag to obtain sufficient abundance. CD82was purified from HEK-293T cells. An integrative proteomic and glycomic approach, which combined glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots, were performed. As a result, we found three N-glycosylation sites and especially Asn157was reported for the first time. Combined lectin blots and LC-MS/MS,27N-glycans structures have been shown for the first time. The presence of some important carbohydrate epitopes provides useful information for understanding and the further investigation of the glycobiological function of CD82.(2) We applied high-sensitive bead-based suspension array system in lectin microarray for the first time. In this system, lectins were conjugated to different populations of microbeads and incubated with biotin-labeled glycoproteins under a liquid condition. Thus, the lectins bound with the specific glycans of glycoproteins under a three-dimensional condition. Our results showed that the bead-based lectin array had the lowest detection of limit among reported lectin microarrays. Furthermore, it provided good linearity with high reproducibility at two to three orders of magnitude in dynamic range. The specific signal patterns for three biotin-labeled glycoproteins were derived with different immobilized lectins, which proved the reliability. Moreover, the quantitative glycosylation alteration of Ig G enriched from sera of healthy individuals and HCC patients was determined. This study demonstrated that the bead-based lectin array can be carried out in a rapid, sensitive, and high-throughput manner and potentially fulfill qualitative and quantitative analyses of clinical samples.(3) The combination of PNGase F mediated incorporation of18O into glycans and tandem MS was applied in the N-glycan structure analysis for the first time. The fragments produced by mass spectrometry contain ambiguous information because fragment ions of the same mass and composition can arise from different regions of the molecule. The glycans labeled with18O at the reducing end were collided by tandem MS. The fragment containing the reducing end, which had an increase of2Da, could be distinguished from the other molecular ions. The increase of2Da using as a reliable marker assisted in the analysis of glycan structure. This approach is simple, convenient for the discrimination of the glycan fragments. On the other hand, HILIC-ESI-MS was used to separate and detect the oliogosaccharides of ovalbumin and human serum Ig G. The positive and negative ion mode was operated. This approach supplied the abundant information for the determination of glycan structure.This thesis consists of four parts which are summarized as follows: Part1. Introduction:a brief and comprehensive introduction of protein glycosylation and glycoproteomics-related fields.Glycosylation is a common and highly diverse co-and post-translational protein modification. It has been reported that more than50%of human proteins are likely conjugated with glycans. There are four major types of protein glycosylation; the two mostly studied are N-linked and O-linked glycosylation. The glycosylation of proteins does not only affect the protein solubility, folding and location but also play an important role in differentiation, development, the metastasis of tumor, fertilization, immunity, infection and regeneration. Therefore, the glycosylation of target glycoprotein should be studied. The part one is emphasis on the study of N-glycosylation including the separation and enrichment of glycoproteins and glycopeptides, the analysis of glycosylation sites, the determination of glycan structure and relative quantification of glycoproteomics and glycomics. We found that mass spectrometry and microarray were complementary technologies for the research of N-glycosylation. Antibody, lectin and glycan microarray are high-throughput, sensitive for the detection of glycosylation. Mass spectrometry exhibits difficulty in discriminating structural isomers and annotating data. Thus, we need new technologies to distinguish the fragment and separate isomers. Generally, glycosylation study now is still full of chances and challenges.Part2. The Study of N-glycosylation Pattern of Recombinant Human CD82(KAI1) by Biological Mass Spectrometry:two sections included (1) determination of CD82N-glycosylation sites;(2) characterization of CD82glycans.(1) CD82is a highly glycosylated membrane protein belonging to the tetraspanins. It has attracted much attention in various human cancers, such as prostate, pancreatic, lung, gastric, hepatic, and colorectal cancers. Although CD82N-glycosylation profoundly affects cell motility and adhesion, its N-glycosylation pattern has not been described so far. Firstly, CD82was expressed in HEK-293T cells with a flag tag to obtain sufficient and purified using anti-FLAG antibody. Secondly, high-performance MS identification and absolute protein expression measurement were conducted to analyze the immnunoprecipited protein. This result confirms that CD82is obtained with very few glycoprotein impurities and with enough quantity as a glycoprotein for the subsequent assay. Thirdly, the presence of the three N-glycosylation sites at the Asn129, Asn157, and Asn198residues was demonstrated using LC-ESI-MS/MS and site-directed mutagenesis. Furthermore, the site-specific oligosaccharides of the two N-glycosylation sites (Asn129, Asnl98) were shown by the digestion of Endo-β-N-acetylglucosaminidase.(2) The characterization of CD82glycan was revealed for the first time. Despite the significant role of carbohydrates, studies on glycosylation have been hindered by its structural heterogeneity and diversity caused by its non-template-driven biosynthesis. In our study, a detailed characterization of recombinant human CD82N-linked glycosylation pattern was conducted by employing an integrative approach, including glycan permethylation, MS analyses and lectin-blot. Lectin-blot is a key approach in glycosylation analysis, due to its ability to recognize specific linkages and distinguish isomeric sugars presented in glycans. Permethylation increases ionization efficiency, produces more structural details in tandem MS and are more suitable for the separation of reversed-phase chromatography. Finally, we found27N-glycans structures of recombinant CD82. The discovered N-glycans could be further classified into three types:high-mannose, hybrid and complex-type. The presence of some important carbohydrate epitopes, such as bisecting GlcNAc, NeuAc, and core fucose, were also detected. The interesting epitopes provide useful information for understanding and the further investigation of the glycobiological function of CD82.Part3. Multiplex Profiling of Glycoproteins Using a Novel Bead-based Lectin Array:two sections included (1) the establishment of bead-based Lectin Array;(2) the application of bead-based Lectin Array.(1) Glycomics is the study of comprehensive structural elucidation and characterization of all glycoforms found in nature and their dynamic spatiotemporal changes that are associated with biological processes. The development of glycomics has been focused on clinical applications, which are viewed as the second golden age of glycomics. Lectin microarray is an attractive platform for structural profile and screening glycosylation differences on large-scale and high-throughput studies. Some glycoproteins have been widely used as disease-specific biomarkers. We applied high-sensitive bead-based suspension array system in lectin microarray to meet the increasing demand of clinical applications. The analytical performance of this approach was evaluated using model glycoproteins, and the results showed that the sensitivity and throughput were improved significantly. Moreover, a good linear response with high reproducibility was obtained. This approach can be further used in the detection of clinical samples.(2) The alteration of glycan structures is a universal hallmark of tumors. Some human serum glycoproteins, such as a-fetoprotein-L3, α1-acid glycoprotein, haptoglobin (Hp), and immunoglobulin G (Ig G), have been widely used as disease-specific biomarkers. Ig G plays an important role in the human immune system, and the attached glycans in Ig G have been implicated in modulating its function. The quantitative glycosylation alteration of Ig G enriched from sera of healthy individuals and HCC patients was determined. Three kinds of glycoproteins were selected as representative glycoproteins to test the reliability of bead-based lectin array. The multiplexed assay provided a high-throughput, sample-and reagent-saving method, allowing for simultaneous detection of multiple carbohydrate epitopes in a single reaction vessel.Part4. The Analysis of N-glycan Chains Using Mass Spectrometry and Hydrophilic Interaction Liquid Chromatography:two sections included (1) the combination of PNGase-mediated incorporation of18O into glycans and tandem MS to analyze the N-glycan structures;(2) the analysis of N-glycan structures using HILIC-ESI-MS.(1) The analysis of N-glycan structure is an important part of glycosylation research. The fragment collided by mass spectrometry are used to deduce the structure of oligosaccharide. However, the fragments produced by mass spectrometry contain ambiguous information because fragment ions of the same mass and composition can arise from different regions of the molecule. Therefore, we combined PNGase-mediated incorporation of18O into glycans and tandem MS to analyze the N-glycan structures. The glycans labeled with18O at the reducing end were collided by MS2. The increase of2Da using as a reliable marker assisted in the analysis of glycan structure.(2) The separation and detection of glycan chains is important step for the analysis of glycan structures. We used HILIC-ESI-MS to analyze the N-glycan structures. HILIC improved the separation and the combination of normal-phase chromatography and mass spectrometry enhance the efficiency of the detection. We analyzed the native N-glycan structures of ovalbumin and human sera Ig G. The negative ion mode was operated for the detection of Ig G sialylation.