基于DIA蛋白质组学技术联合PRM验证探讨去卵巢骨质疏松大鼠差异蛋白表达

Differential Protein Expression in Ovariectomized Rat Model of Osteoporosis Based on DIA Proteomics and PRM Verification

目的:基于非数据依赖性采集(DIA)蛋白质组学联合平行反应监测(PRM)靶向质谱技术筛选去卵巢大鼠股骨组织的差异蛋白,寻找其中可能与骨质疏松(OP)发病相关的关键靶蛋白。方法:Wistar雌性大鼠16只,随机分为正常对照组和模型对照组各8只,模型对照组采用卵巢切除法建立骨质疏松症模型。12 w后双能X线骨密度仪测量大鼠全身骨密度(BDM);解剖分离股骨标本进行Micro-CT扫描和重建;对正常对照组和模型对照组大鼠股骨组织进行蛋白DIA定量分析,筛选差异蛋白并进行生物信息学分析,并采用PRM技术验证两组间差异蛋白。结果:造模12 w后,与正常对照组比较,模型对照组骨小梁数量(Tb.N)、骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、连接密度(Conn.D)均明显下降(P<0.05或P<0.01),骨小梁分离度(Tb.Sp)、结构模型指数(SMI)显著升高(P<0.01);Micro-CT三维重建可见骨小梁整体结构退变、体积减少、连接断裂;DIA检测鉴定出显著差异蛋白234个,其中上调蛋白162个,下调蛋白72个,KEGG通路富集显示这些差异蛋白主要参与粘着斑、RNA转运、TGF-β、等信号通路。对关键差异蛋白进行PRM建库分析,检测到27个候选蛋白,其中双特异性丝裂原活化蛋白激酶1(MAP2K1)、谷胱甘肽过氧化物酶4(GPX4)、丝裂原活化蛋白激酶1(MAPK1)、软骨寡聚基质蛋白(COMP)、等11个蛋白定量分析明显具有差异(P<0.05或P<0.01)。结论:OP大鼠股骨组织内有大量蛋白质差异表达,MAP2K1、GPX4、MAPK1、COMP等11个差异蛋白可能与骨质疏松发病密切相关。

Objective:To screen out the differential proteins in the femur of ovariectomized rats based on data-independent acquisition(DIA)proteomics and parallel response monitoring(PRM)-based targeted mass spectrometry,and to find the key target proteins that may be involved in the pathogenesis of osteoporosis(OP).Methods:Sixteen female Wistar rats were randomized into a blank group(n=8)and a model group(n=8).The model group was ovariectomized to establish the OP model.After 12 weeks,the whole body bone mineral density(BDM)of rats was measured by dual energy X-ray absorptiometry,and the dissected femur specimens were scanned and reconstructed by Micro-CT.The data from DIA proteomics of the normal group and model group was quantitatively analyzed,and the differential proteins between the two groups were screened out by bioinformatics tools and verified by PRM.Results:Twelve weeks after modeling,compared with the blank group,the model group showed decreases in trabecular number(Tb.N),bone volume fraction(BV/TV),trabecular thickness(Tb.Th),and connection density(Conn.D)(P<0.01 or P<0.05)and increases in the trabecular separation(Tb.Sp)and structural model index(SMI)(P<0.01).Micro-CT three-dimensional reconstruction showed structural degeneration,volume reduction,and junction breakage of bone trabeculae.A total of 234 significantly differential proteins were identified by DIA,including 162 up-regulated proteins and 72 down-regulated proteins.KEGG pathway enrichment showed that these differential proteins were mainly involved in adhesion plaque,RNA transport,TGF-βand other signaling pathways.PRM database analysis yielded 27 candidate proteins,among which 11 proteins such as dual specificity mitogen-activated protein kinase kinase 1(MAP2K1),glutathione peroxidase 4(GPX4),mitogen-activated protein kinase 1(MAPK1),and cartilage oligomeric matrix protein(COMP)showed significantly different expression(P<0.01 or P<0.05).Conclusion:There are a large number of differential proteins in the femur of OP rats.Eleven differential proteins such as MAP2K1,GPX4,MAPK1,and COMP may be associated with the pathogenesis of OP.