The Proteomics Study About Serum Of Patients With Ankylosing Spondylitis By TMT And LC-MS/MS

Background:Ankylosing spondylitis(AS)is a autoimmune and inflammatory diseases,with chronic and progressive characteristics,the pathological characteristic is chronic inflammation of tendon and ligament attachment point,mainly involving the sacroiliac joint,spine bone protrusion,paravertebral soft tissue,tendon and soft tissue and peripheral joints.And may be associated with articular manifestations;early diagnosis,early treatment,help to improve the prognosis of patients with AS,improve the quality of life of patients,reduce the rate of disability.Because the cause of the disease is not clear,most studies suggest that the pathogenesis of AS may be related to heredity,infection,immunity and other factors.This study is through the method of proteomics,using tandem mass spectrometry(TMT label)combined with tandem mass spectrometry,liquid chromatography analysis of the serum of AS patients,screening and identification of differentially expressed proteins relative to healthy adults and patients with diffuse idiopathic hyperostosis(DISH).Objective:To explore the specific differences in pathology AS protein,serum protein markers for early diagnosis of this disease,and to further explore the cause of disease,to explore ways and drugs for early clinical diagnosis and therapeutic target and provide a basis for the evaluation of the prognosis of AS and susceptible population screening.1)In accordance with the diagnostic criteria of AS in ESSG(1991)and axial SPA(Spondyloarthritis,SPA)in ASAS(2009),6 cases of AS,6 cases of DISH(according to Resnick diagnostic criteria)and 6 cases of normal persons were collected.4°C low temperature centrifugation,the upper serum,and packaging logo,and stored in-80°C at all;serum removal of high abundance protein,SDS-PAGE,then proteins were digested with trypsin.Trypsin get peptide protein digested and labeled with TMT,TMT6-127 markup for the control group,TMT6-129 marker for AS group,TMT6-131 DISH group for marking,the peptides labeled mixed After the treatment,LC-MS /MS was detected by Dionex Ultimate 3000 NL chromatograph combined with Q-Exactive mass spectrometer,except for salt and impurity removal.The SEQUEST HT retrieval algorithm is used to retrieve the Swissprot Human database by spectral graph.The search software is Proteome Discoverer,and the data is analyzed by Xcalibur 2.1.2 software.According to the relative abundance of ions reported by different TMT reagents,the relative quantitation of peptides was carried out,and the relative quantitative results of protein were quantified according to the relative quantitative results of peptides.The ratio of the same protein in a certain group to the other group(Ratio)is considered to be more than 1.5 times the ratio determined,that is,the increase is greater than 1.5 or less than 0.75,which is considered a significant difference.2)We will make preliminary bioinformatics analysis of all the identified differentially expressed proteins,and analyze the mechanisms of the differential proteins,so as to predict the relationship between the differential proteins in the biological network and further screen the meaningful proteins.Results:This study identified 424 proteins,which reached the relative number of protein standards for 144,two and above were quantified using specific peptide protein number 61,the number of protein was quantified using a specific peptide is 15,the relative health control group has a total of 22 up-regulated protein and 42 downregulated proteins;relative to the DISH group has a total of 21 up-regulated proteins,24 down regulated proteins.Conclusions:1.According to the TMT marker protein quantitation,and liquid chromatography tandem mass spectrometry analysis of the serum of patients with AS in comparison of healthy control group and DISH group of differential proteomics research,explore and establish AS serum TMT of LC-MS-MS.2.The specific serum markers and pathogenesis of AS were explored,and the differential proteome database of AS patients was established.The results showed that there were significant changes in the serum of patients with AS compared with normal subjects and DISH group.3.Through this study,we based on the proteomics research on the screening of protein bioinformatics analysis,trying to find some potential biomarkers of AS,to lay the foundation for further study.