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 investigate the serum protein targets of Qianggu Decoction(强骨饮, QGD) on treating osteoporosis by the proteomics analysis using tandem mass tag(TMT) and liquid chromatographytandem mass spectrometry(LC-MS/MS). Methods: Twenty serum protein samples were recruited(10 patients with primary type Ⅰ osteoporosis before and after QGD treatment) and the high abundance ratios protein was removed, two serum samples were extracted and labeled with TMT reagent. Then, mass spectrometric detection, identification of differentially expressed proteins and bioinformatics analysis of differentially expressed proteins were carried out. Results: A total of 60 proteins were identified, within a 99% confidence interval, to be differentially regulated of which, 34 proteins were up-regulated and 26 proteins were down-regulated. Differentially expressed proteins analyzed by Gene Ontology(GO) annotation mainly get involved in 12 different biological processes, 7 types of cellular components, and 6 kinds of molecular functions. Angiotensinogen(AGT), stromelysin-1(MMP3), heparanase(HPSE) and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were screened as candidate protein targets of QGD treatment, which were related to metabolic mechanism of bone remodeling and/or bone collagen of osteoporosis. By the utilization of the protein-protein interaction network analysis tool named STRING10.0, it showed that AGT, MMP3, HPSE and GAPDH were located in the key node of the protein-protein interactions network. Furthermore, AGT, MMP3, HPSE and GAPDH were found to be directly related to BMP, MAPK, Wnt, SMAD and tumor necrosis factor ligand superfamily member 11(TNFSF11) families. Conclusions: The proteomics analysis by using TMT combined with LC-MS/MS was a feasible method for screening the potential therapeutic targets associated with QGD treatment. It suggests that AGT, MMP3, HPSE and GAPDH may be candidate protein targets of QGD treatment which can be used as therapeutic effect monitor and early diagnosis of primary type Ⅰ ost
