Mass Spectrometry-based Methodology For Quantitative Analysis Of Serum Protein/Glycoprotein In Diabetes Mellitus

The human body is an organism in dynamic change,and all kinds of reactions are carried out at every moment.In order to have a deep understanding of ourselves,human beings have been devoted to the study of gene level,protein level and metabolic level;The study of life system from protein level provides important insights into many life processes.Proteomics is a science that studies all proteins and their dynamic changes in cells.It studies organisms quantitatively,dynamically and holistically at the protein level,and thus obtains a comprehensive and complete understanding of disease processes,cellular physiological and biochemical processes and regulatory networks at a deeper level.Glycoproteomics is based on proteomics.Glycosylation modification of proteins is one of the most common and important post-translational modifications of proteins.It participates in many biological processes,and abnormal glycosylation modification of proteins has been found to be associated with the pathological process of major diseases such as tumors and neurodegenerative diseases.At present,many clinical biomarkers are glycoproteins.However,the abundance of glycoprotein/glycopeptide in complex biological samples is low and the ionization efficiency of glycopeptide is low and easily inhibited by other non-glycopeptides.Therefore,the enrichment of glycoprotein/glycopeptide before mass spectrometry analysis is a crucial step in the process of glycoprotein analysis.Mass spectrometry-based proteomics/glycoproteinomics has greatly changed the way in which biological systems are questioned;It can measure thousands of proteins and post-translational modifications simultaneously,making it possible to study complex biological processes ranging from protein complexes to human patient populations.In the second chapter,Label-free technique was used to quantify proteins in the protein level and glycosylation level.6 normal serum samples and 6 diabetic serum samples were analyzed.291 non-redundant proteins and 181 non-redundant glycoproteins were obtained.There were 65 proteins and 24 glycoproteins with significant difference between the disease group and the control group.KEGG pathway analysis showed that differential glycoproteins were significantly directed to diabetes mellitus.These glycoproteins were widely involved in complement and coagulation cascade pathways,and were closely related to the occurrence and development of diabetes mellitus.In biomedical research,disease-related proteins,which can be used as biomarkers,are generally of low and medium abundance.In the third chapter,we use the high abundance column to remove the first 14 proteins with the highest abundance in serum,and verify the effect of high abundance removal by SDS-PAGE experiment.Trypsin/Lys-c combination is relatively simple and saves time,and it greatly increases the number of protein of detection in the experiment.In order to detect more proteins,the mixed peptide segments were fractionated offline after iTRAQ labeling of enzymatically hydrolyzed peptide segments.The mass spectrometry data were quantified to 418 proteins by bioinformatics analysis.There were 14 proteins with significant difference between the disease group and the control group.Through the analysis of 14 differential proteins,it was found that the differential proteins in the adipose digestion and absorption pathway were mainly related to the adipose absorption process and fatty acid transport,which directly or indirectly affected the pathogenesis of diabetes.In summary,we conducted quantitative studies of diabetes through two different quantitative strategies to systematically and comprehensively reveal changes in protein and glycosylation modifications during the onset of diabetes.This series of proteins that are differentially expressed at the protein level and level of glycosylation provides a detailed data reference for studying the development of diabetes.Quantitative studies of proteins in diabetic human serum are useful to further reveal the pathogenesis of diabetes.