Development Of New Approaches For Glycoproteomics And The Application In The Study Of IgA Nephropathy

Glycosylation is one of the most complex and important protein translational modifications and is involved in many biological processes such as gene transcription,protein translation,signaling,cell-cell and host-pathogen interactions.The most commom glycosylations include N-glycosylation,O-GalNAc glycosylation and O-GlcNAc glycosylation,which have various structures and widely distributed.Abnormal changes in protein glycosylation is related to the occurrence and development of many important diseases,including immune diseases,cancer,congenital sugar defects and so on.Hence,the study on protein glycosylation has drawn the more and more attention of researchers recently.This thesis mainly focuses on the investigation and application of new technique for human plasma glycoproteomics,including the in-depth qualitative and quantitative analysis of the N-glycans and O-glycopeptides of plasma IgA1 from healthyl controls,Non-CreIgA nephropathy and CreIgA nephropathy patients,systematic analysis of their plasma O-glycoproteome as well as the development and application of new materials based on C₃N₄ matrix for the high efficiency enrichment of glycopeptides.The thesis consists of four parts.Chapter one:We summarized the basic concepts,research methods and biological functions of protein glycosylation.Besides,we also reviewed IgA1glycosylatio in IgA nephropathy,the difficulties of glycosylation research in methodology and the status of researches on glycoproteome including the serum O-glycoproteome,enrichment methods of glycopeptide.Chapter two:IgA nephropathy?IgAN?is a common primary glomerular disease in the world.The IgA1 deposition in the glomerular mesangial area and persistent inflammation are typical immunopathological features of this disease.Crescent IgA nephropathy is one serious type.Previous studies have shown that IgA1 in the plasma of IgAN patients undergoes abnormal glycosylation,but the changes of glycosylation of IgA1 has never been systematically investigated before.Here,we have established a simple,rapid and reproducible method for sample preparation and mass spectrometry analysis firstly.Then the N-glycans and intact O-glycopeptides of mIgA1 and pIgA1 were successfully quantified and compared among 12 healthy controls?HC?,11 non-crescentic IgA nephropathy patients?Non-CreIgAN?and 11crescentic IgA nephropathy patients?CreIgAN?.We found that some of the glycans?mannose?were differentially expressed in mIgA1 and p IgA1,such as 4 N-glycans and 13 intact O-glycosylated peptides of mIgA1 were significantly differently?P<0.01?expressed in three sets of samples,4 N-glycans and 17 intact O-glycosylated peptides of pIgA1 were significantly differently expressed in three sets of samples?P<0.01?.These N-glycans and intact O-glycosylated peptides may serve as potential disease diagnostic markers or therapeutic targets,and provide basis for classifying and explaining the pathogenesis of IgA nephropathy.Chapter Three:The altered O-glycoproteome is associated with disease pathological state including cancer,inflammatory and degenerative diseases.In order to discover abnormally expressed O-glycoproteins/peptides in plasma of patients with IgA nephropathy and as potential biomarkers for the classifying and diagnosis of IgA nephropathy,we conducted an O-glycoproteomics research.Because of the microheterogeneity and macroheterogeneity of O-glycosylation,site-specific O-glycosylation analysis in human plasma is still challenging.Here,we developed a systematic strategy that combined multiple enzyme digestion,multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization.We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThc D is not only suitable for the identification of singly O-glycosylated peptides?50.3%?but also doubly?21.2%?and triply?28.5%?O-glycosylated peptides.Totally,with the strict scoring criteria,499 non-redundant intact O-glycopeptides,173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human plasma,including 121 novel O-glycosylation sites.C urrently,this is the largest data set of site-specific O-glycoproteome from human plasma samples.We expect that the strategies developed by this study will facilitate in-depth analyses of O-glycoproteomes in human plasma and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases?IgA nephropathy?.Besides,Stable isotope dimethyl labeling multiplex quantitative O-glycoproteomics method was developped and applied to discover important O-glycoproteins in IgAN patients.The quantative results can determine the relative changes in the level of O-glycoproteins,which potentially playing important roles in certain diseases or disease states and can be used as bimarker.Chapter Four:Due to the low stoichiometry of glycopeptides in proteins,the enrichment of glycopeptides from complex biological samples is crucial for in-deepth coverage of glycoproteins.In the previous two studies,The specificity and enrichment efficiency of commercial HILIC are not enough high.Therefore,we need to develop a glycopeptide enrichment material with high specificity and high enrichment efficiency.Herein,we present a facile and economical procedure to generate a new self-assembling phenylboronic acid functionalized straticulate C₃N₄-based hydrophilic material?MPBA-Au@s-C₃N₄,MASC?,with enhanced affinity capability towards glycopeptides based on the synergetic effect,provided by the hydrophilic property and B–N coordination of the material.XPS spectra,Zeta potential tests and the Alizarin Red S?ARS?color reaction of the as-prepared materials proved the existence of B-N bonds.The performance of this material was evaluated using standard protein IgG and horseradish peroxidase?HRP?.The results show that the material possess excellent properties,including lo w pH value adaptation,high hydrophilicity and stability,good repeatability and recyclability,high selectivity?1:100?,low limit of detection?0.33 fmol/?L?,high enrichment efficiency?⁸0%?and high recovery rate?⁹0%?.The materials were successfully applied to capture glycopeptides in an unbiased manner from standard pro teins and complex samples.As demonstrated that,37 glycopeptides from IgG and 21 glycopeptides from horseradish peroxidase?HRP?were identified.The performance of MASC on human urine and plasma glycoproteome analysis was also tested.An average of 1465 glycopeptides from 839glycoproteins and 1553 glycopeptides from 884 glycoproteins were identified from female and male urine samples in a single mass spectrometry analysis.Additionally,463 glycopeptides assigned to 209 glycoproteins were identified from 5?L of human plasma.It is proved that MASC can effectively enrich glycopeptides from human blood and urine,and it may be used for finding glycopeptide markers in diseases such as IgA nephropathy.It has a good application value in the research of glycoproteomics..