膝骨性关节炎差异表达蛋白血清标记物筛选:iTRAQ技术的重要性

iTRAQ technology is important to screen serum markers of differentially expressed proteins in knee osteoarthritis

背景:在临床上对于早期膝关节骨性关节炎尚缺乏有效的诊断方式。蛋白质组学是从整体的角度分析细胞的所有蛋白质组成、表达水平及修饰状态的学科,可以了解蛋白与蛋白间的相互作用及联系,从而揭示蛋白功能与细胞生命活动的规律。目的:通过对骨性关节炎K/L分级的各阶段的血清样本进行ITRAQ定量蛋白组学分析检测,以发现骨性关节炎各个时期的的潜在分子标志物。方法:收集膝关节骨性关节炎患者60例,参照Kellgren-Lawrence(K-L)分级标准,分为K-L 0、Ⅱ、Ⅳ级的亚组,每个亚组随机选取10例男性与10例女性。去除血清高丰度蛋白,进行稳定同位素116、117、118的iT RAQ标记、反相色谱柱分离、质谱检测及Swissport数据库检索;再利用生物信息学软件进行分析。结果与结论:通过对不同样品的iT RAQ肽段实验标记、Q-star质谱鉴定和MASCOT搜库,共筛选出有意义差异蛋白169个,K-L 0级与K-LⅣ级差异蛋白153个;K-L 0级与K-LⅡ级差异蛋白153个;K-LⅡ级与K-LⅣ级差异蛋白145个。结果表明应用iT RAQ技术可筛选出与骨性关节炎发生发展的多个差异蛋白,提示iT RAQ技术对于骨性关节炎蛋白质组学血清生物标记物的研究有重要意义。

BACKGROUND;There is lack of effective diagnostic methods for early knee osteoarthritis.Proteomics refers to the large-scale experimental analysis of proteomes from the overall level of intracellular protein compositioon,expression and modification,based on which,we can understand protein-protein interaction and relationship,thereby revealing protein functions and cell activity patterns.OBJECTIVE;To apply the isobaric tags for relative and absolute quantitation（i TRAQ） to identify proteins differentially in serums of osteoarthritis Kellgren-Lawrence classification at each stage,and to find out potential molecular markers at each stage of osteoarthritis.METHODS;Sixty patients with knee osteoarthritis were enrolled,and according to Kellgren-Lawrence（K-L） grading,they were subdivided into K-L 0,II and IV groups.There were 10 males and 10 females randomly selected in each group.Proteins differentially regulated in serums were identified by the stable isotopes 116,117,118 of the i TRAQ labeled,reversed-phase column separation,mass spectrometry and Swissport database search,after the serums were subjected to high abundance proteins depletion.Finally,we analyzed the proteins identified by using bioinformatics software.RESULTS AND CONCLUSION;Totally 169 proteins were identified through i TRAQ peptides experiments of different sample tags,Q-star mass spectrum identification and MASCOT library search.153 proteins were identified as K-L 0 to K-L IV,153 proteins were identified as K-L 0 to K-L II,and 145 proteins were identified as K-L II to K-L IV.i TRAQ technology may help to identify novel serum markers for early diagnosis of knee osteoarthritis,indicating i TRAQ technology for proteomics serum biomarkers of osteoarthritis study has good application prospect.