The Proteomics Study About Serum Of Patients With Diffuse Idiopathic Skeletal Hyperostosis By TMT And LC-MS-MS

Background: DISH is characterized by calcification and ossification of soft tissues, mainly ligaments and entheses. The diagnosis of DISH is currently based on classification criteria that require the involvement of the spinal thoracic segment on conventional radiographs. Although prevalence estimates vary with the number of bridges used to define the disease, the prevalence of DISH is consistently high and increases with age and obesity. But the early diagnosis of disease is difficult to be confirmed due to the insufficient imaging finding. And the research into the pathophysiology of DISH has not established any specific biomarkers of serum levels. Therefore, it is very important to ascertain the etiology of DISH and establish related biomarkers which can diagnose and predict the DISH. The development of proteomics leads to a new period for the basic research of the pathophysiology of DISH. The recently introduced technology of Tandem Mass Tag(TMT) and Liquid Chromatography-tandem mass spectrometry(LC-MS/MS) is believed to be a highly reliable and accurate tool for quantitative analysis of differentially expressed proteins.Objective: 1. To detect and identify the differently expressed proteins in serum between DISH patients and normal controls by the technology of TMT and LC-MS-MS. 2. To analyze the function and role of the selected differential proteins which in the pathogenesis of DISH by knowledge of the bioinformatics. 3. To predict and screen the potential possible biomarker of patients of DISH.Methods: 1) The serums were collected from 10 DISH patients and 10 normal volunteers whom with the same distribution of age and sex. There’s no age or sex differences between two groups. After removing the high abundance ratios protein and SDS-PAGE, the samples were digested by trypsin and labeled with TMT reagents(131 for the DISH group, 127 for the normal control group). group). And then a Liquid Chromatography-tandem mass spectrometry analysis(LC-MS/MS) was performed and the differential expressed proteins were identified after network　database research according the premade standard. 2) The differential proteins were filtrated by software and manual analysis. And then bioinformatics analysis was performed to analyze the role and relationship of the candidate proteins in the biological network and predict and screen the potential possible biomarker of patients of DISH.Results: In our quantitative proteomics analysis, 425 human proteins were identified using TMT labeling in conjunction with LC-MS/MS. And 253 proteins were deem to be significative because of which scores greater than 5. Among these significative proteins, the differential proteins with up-regulation greater than 1.3 times was in 17, and which with down-regulation smaller than 75% was in 56. And another two proteins only can be detected from DISH group which are Ig alpha-2 chain C region and Isoform H14 of Myeloperoxidase(MPO). And the KEGG bioinformatics analysis show that extracellular superoxide dismutase(Cu-Zn), glutathione peroxidase(GPx3), PLTP, APO-AI and APO-AII can be found in signal path PPAR. And we analyzed that MPO also maybe play the role in the upstream or downstream of PPAR and Wnt.Conclusions: 1. This is the first time to study comparative serum proteomes and explore the technical route of TMT and LC-MS-MS of DISH. 2. By this study, we created a database of serum differential proteins of DISH, and identifed some significative and quantitative proteins and two particular proteins which only appeared in DISH successfully. 3. Base on the bioinformatics analysis, we found that several proteins take part in the biological activity of path PPAR and Wnt. So that these proteins maybe the potential serous biomarker of DISH, and the pathogenesis of DISH can be studied further by these proteins in the future.