CAJ | 학술논문

目的应用串联质量标签(tandem mass tag,TMT)标记联合高效液相色谱-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,经去除血清高丰度蛋白、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;初步筛选出与骨质疏松骨重建相关的RAB7A、TSP1、GAS6、SPP24共4个候选蛋白.通过STRING10.0蛋白相互作用网络分析工具发现RAB7A、TSP1、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、TSP1、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、TSP1、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.
목적 응용천련질량표첨(tandem mass tag,TMT)표기연합고효액상색보-질보/질보법(LCESI-MS/MS)기술감정절경후골질소송환자화절경후골량정상부녀혈청차이단백질,사사절경후골질소송증적혈청단백분자표지물.방법 회고성분석2013년3월지2014년3월초차진단10례절경후골질소송부녀급10례절경후골량정상부녀자료,분별획취혈청,경거제혈청고봉도단백、측정단백농도、SDS-PAGE단백제취측정화TMT표첨표기등과정후,진행고효액상천련질보태단검측,Mascot search engine연건수색SwissProt단백질수거고감정다태,사선차이표체적단백,진행생물신식학분석.결과 종경TMT표기연합LC-ESI-MS/MS기술감정적단백질표체보중공사선출차이표체단백87개(상조단백50개,하조단백37개).대차이표체단백진행GO기인주석분석,발현기주요삼여15충생물학과정,주요래자7충세포조성,주요구유6충분자공능;초보사선출여골질소송골중건상관적RAB7A、TSP1、GAS6、SPP24공4개후선단백.통과STRING10.0단백상호작용망락분석공구발현RAB7A、TSP1、GAS6재상호작용망락도중처우공능망락교차점;이SPP24처우망락변연,단시기여골중건중요단백BMP-2직접상관.저사차이표체적단백RAB7A、TSP1、GAS6、SPP24여절경후골질소송증적발병궤제유관.결론 본연구증명기우TMT표기연합LC-ESI-MS/MS기술가작위감정사선절경후골질소송증혈청단백분자표지물적유효방법;RAB7A、TSP1、GAS6、SPP24가능시절경후골질소송증적잠재유효혈청분자표지물.
Objective To screen the serum protein molecular markers of postmenopausal osteoporosis by the proteomicsanalysis 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 postmenopausal patients with osteoporosis and 10 cases of postmenopausal 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 differentiated 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 involved in 15 kinds of biological processes,7 kinds of cellular component,6 kinds of molecular function.RAB7A,TSP1,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,TSP1,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-ES1-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 postmenopausal osteoporosis.