Screening Of Serum Differential Proteins Related To Exercise Fatigue And Isolation, Enrichment And Detection Of Glycoproteins

With the implementation and propulsion of the human genome project,life science research has entered the post genome era.In this time,the main research object of life science is the functional genomics,including structural genome and proteome research.Proteome is a kind of subject which is related to proteome corresponding to genome and research more protein system,in particular of interaction among many different proteins in a large system.Exercise fatigue is a nonspecific complex physical phenomenon.The mechanism and elimination methods of exercise-induced fatigue have been paid attention to by experts and scholars from all over the world,but the judgment of exercise-induced fatigue was less researched.The body fatigue was reflected mainly through measuring some biological indicators related to the stress response,inflammation factors and the change of metabolite,such as cortisol,testosterone,blood sugar,glycogen,blood lactate and blood urea nitrogen.Exercise fatigue process,however,involves a wide variety of metabolites,which are affected by the form,intensity and time of exercise.The exercise for a long time can result in the fatigue,raising the ratio of protein power,which caused the different degree of damage to skeletal muscle.So we speculated that the degree of exercise-induced fatigue should have been judged and detected by knowing the change of protein metabolism.Therefore,we compared plasma proteomic of the normal state to the exercise-induced fatigue individuals through the proteomics technology and identified 12 abnormal proteins using two-dimensional electrophoresis combined mass spectrometry technology,respectively including 6 up-regulated and 6 down-regulated proteins after the exercise-induced fatigue.The down-regulated proteins covered alpha-1B-glycoprotein,clusterin,Ig a-1 chain C region,angiotensinogen,complement C4-A and haptoglobin.The up-regulated proteins included alpha-2-HS-glycoprotein,antithrombin-?,serum albumin,CD5 antigen-Like,haptoglobin-related protein and apolipoprotein E.These expression different proteins were involved in acute phase proteins,immune-related proteins and lipid metabolism-related proteins and neuromo-dulation-related proteins.However,protein was the main executor of the organism function.The protein glycosylation was one of the important protein post-translational modification and more than 50%proteins possessed sugar chains in cells.The sugar chain of glycoprotein like clothes was on the cell surface and constituted the antenna and the tentacles of cells.In recent years,with the rapid development of proteomics,glycoproteomics also arises at the moment,and gradually became another new frontiers and hot spots in the research field of analytical chemistry and life science.Glycoprotein in biological system,however,was low absolute abundance and entrained,coated and masked by high abundance ratios of non-glycoprotein,which made the most of glycoproteins were difficult to be extensively applied in the field of medicine.Now,how to remove the high abundance ratios of non-glycoprotein,search and enrich the low abundance of glycoproteins,has become one of the important issues to glycoproteomics research.Therefore,we successfully prepared the boric acid functionalized modifications of different materials for the separation and enrichment of glycopeptide or glycoprotein,and attempted to establish the detection method of glycoprotein on the basis of the quantum dots as fluorescent probesin,which was on the basis of cross subjects including biology,chemistry and materials science as the background.These studies of glycoproteins provided a certain reference value to the research of biological sciences,medicine and exercise physiology.At first,SiO₂@Fe₃O₄ magnetic microspheres were synthesized using ultrasonic method,which saved more time than traditional mechanical mixing method.Then phenylboronic acid functional SiO₂-APBA@Fe₃O₄ microspheres were successfully prepared through grafting 3-aminophenylboronic acid on the surface of Si02@Fe₃O₄ microsphere.The glycoproteins/glycopeptides were selectively separated and enriched on the basis of the reversible affinity between glycoprotein and phenylboronic acid groups on the surface of SiO₂-APBA@Fe₃O₄ microsphere under different conditions of acid and alkaline.The results were that SiO₂-APBA@Fe₃O₄ microspheres had obvious selective enrichment for glycoprotein/glycopeptide,and had a high adsorption capacity and adsorption efficiency,which indicated SiO₂-APBA@Fe₃O₄ microspheres possessed the potential role of the separation and enrichment of glycoprotein/glycopeptide from complex biological samples.The second,phenylboronic acid functional PGMA/EDMA?APBA@PGMA/EDMA?microspheres were successfully synthesized.Then we established the SPE method for the enrichment of glycoprotein/glycopeptide using the APBA@PGMA/EDMA microspheres as the stationary phase.The results showed that the APBA@PGMA/EDMA microspheres had the obvious adsorption selectivity and high efficiency to glycoproteins/glycopeptide.In addition,the both N-linked glycosylation peptide and O-linked glycosylation peptide could be separated from the human serum samples through the SPE method using the APBA@PGMA/EDMA microspheres as stationary phase.The result showed that the method had potential application value of capturing the glycoprotein from the complex biological samples.Finally,a new method of glycoprotein detection was set up based on the QDs,which provided a new way of thinking to promote the application of QDs as fluorescent probes in biological analysis.