基于TMT标记联合LC-ESI-MS／MS技术筛选绝经后骨质疏松症的血清分子标志物研究  

Serum protein biomarkers of postmenopausal osteoporosis based on TMT labeling and LC-MS/MS technology

作　　者：史晓林[1] 梁博程[2] 姚建亮[2] 李春雯[2] 施振宇[2] 李旭云[2] 

机构地区：[1]浙江中医药大学第二附属医院骨伤科,杭州310005 [2]浙江中医药大学

出　　处：《中华骨科杂志》2015年第10期1004-1010,共7页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金面上项目（81373878）;浙江省自然科学基金项目（LY13H290009,LY12H29007）

摘　　要：目的应用串联质量标签（tandem mass tag，TMT）标记联合高效液相色谱一质谱／质谱法（LCESI-MS／MS）技术鉴定绝经后骨质疏松患者和绝经后骨量正常妇女血清差异蛋白质，筛查绝经后骨质疏松症的血清蛋白分子标志物。方法回顾性分析2013年3月至2014年3月初次诊断10例绝经后骨质疏松妇女及10例绝经后骨量正常妇女资料，分别获取血清，经去除m清高丰度蛋白、测定蛋白浓度、SDS-PAGE蛋白提取测定和TMT标签标记等过程后，进行高效液相串联质谱肽段检测，Mascot search engine软件搜索SwissProt蛋白质数据库鉴定多肽，筛选差异表达的蛋白，进行生物信息学分析。结果从经TMT标记联合LC-ESI-MS／MS技术鉴定的蛋白质表达谱中共筛选出差异表达蛋白87个（上调蛋白50个，下调蛋白37个）。对差异表达蛋白进行GO基因注释分析，发现其主要参与15种生物学过程，主要来自7种细胞组成，主要具有6种分子功能；初步筛选H1与骨质疏松骨重建相关的RAB7A、TSPI、GAS6、SPP24共4个候选蛋白。通过STRING10．0蛋白相互作用网络分析T具发现RAB7A、TSPI、GAS6在相互作用网络图中处于功能网络交叉点；而SPP24处于网络边缘，但是其与骨重建重要蛋白BMP-2直接相关。这些差异表达的蛋白RAB7A、TSPl、GAS6、SPP24与绝经后骨质疏松症的发病机制有关。结论本研究证明基于TMT标记联合LC-ESI-MS／MS技术可作为鉴定筛选绝经后骨质疏松症血清蛋白分子标志物的有效方法；RAB7A、TSPl、GAS6、SPP24可能是绝经后骨质疏松症的潜在有效血清分子标志物。Objective To screen the serum protein molecular markers of postmenopausal osteoporosis by the proteomics- analysis using Tandem Mass Tag （TMT） combined with liquid chromatography-tandem mass spectrometry （LC- ESI- MS/MS）. Methods 20 serum protein samples were recruited （10 cases of postmeuopausal patients with osteoporosis and 10 cases of post- menopausal women without osteoporosis）and the high abundance ratios protein was removed, differentiation protein was extracted and labeled with TMT reagent. Then, mass spectrometric detection, data analysis of differentially expressed proteins, and analysis of biological information were carried out. Results 87 significantly differentially expressed proteins were screened from the dif- ferentiated protein expression profile by LC-ESI-MS/MS combined with TMT labeling. While 50 proteins were up-regulated, and 37 proteins were down-regulated. Differentially expressed proteins were analyzed by GO annotation, these proteins are mainly in- volved in 15 kinds of biological processes, 7 kinds of cellular component, 6 kinds of molecular function. RAB7A, TSPI, GAS6, SPP24 were screenedas candidate proteins which were related to mechanism of bone remodeling of osteoporosis. By STRING 10.0 protein interaction network analysis tools, RAB7A, TSPI, GAS6 were located in the center of the interaction network. SPP24 was located at edge of the network, but it is directly related to the protein BMP-2 of bone remodeling. RAB7A, TSP1, GAS6, SPP24 may be associated with the pathogenesis of postmenopausal osteoporosis. Conclusion These results provide that the proteomics analysis by using TMT coupled with LC-ESI-MS/MS was a feasible method for screening the molecular biomarkers. It suggests that RAB7A, TSP1, GAS6 and SPP24 may be useful biomarkers which can be used both in diagnosis and treatment of postmeno- pausal osteoporosis.

关 键 词：骨质疏松 绝经后 骨重建 生物学标记 蛋白质组学 

分 类 号：R580[医药卫生—内分泌]