微量样本蛋白质组前处理流程的建立及衰老小鼠单个窦卵泡蛋白质组图谱的绘制  

Development of a low input sample proteomics preprocessing protocol and mapping aging atlas of mouse antral follicles at single follicle resolution

作　　者：张新帅 李红超 付斌 贺福初 王晓文 李杨 ZHANG Xinshuai;LI Hongchao;FU Bin;HE Fuchu;WANG Xiaowen;LI Yang(State Key Laboratory of Medical Proteomics,Academy of Military Medical Sciences,Beijing Proteome Research Center,National Center for Protein Sciences(Beijing),Beijing 102206,China;Research Unit of Proteomics-Driven Cancer Precision Medicine,Chinese Academy of Medical Sciences,Beijing 102206,China)

机构地区：[1]军事医学研究院,医学蛋白质组全国重点实验室,北京蛋白质组研究中心,国家蛋白质科学中心(北京),北京102206 [2]中国医学科学院蛋白质组学驱动的肿瘤精准医学创新单元,北京102206

出　　处：《中国药理学与毒理学杂志》2024年第12期917-931,共15页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(32088101);国家自然科学基金(31900666);国家重点研发计划(2021YFA1301603);蛋白质组学国家重点实验室自主研究课题(SKLP-Y202204)。

摘　　要：目的建立一种针对微量样本(1×10^(4)细胞)的蛋白质组前处理流程,用于衰老小鼠单个窦卵泡的蛋白质组图谱绘制,并对窦卵泡的衰老图谱进行解析。方法利用293T细胞,通过对比不同的裂解液、酶切条件、超声处理方式等,建立针对1×10^(4)数量级细胞样本(约1μg数量级蛋白质)的蛋白质组前处理流程。通过与商业化的样本前处理试剂盒(iST试剂盒和Ex极微量试剂盒)进行对比,从蛋白质和肽段鉴定重叠性、蛋白质整体亲/疏水性、蛋白质理论等电点以及蛋白质分子质量4方面对本研究所建立的样本前处理流程进行评估。以100,1000和100003种数量级小鼠原代脾和肝细胞样本对样本前处理流程的样本适用性进行评估。基于该样本前处理流程,结合timsTOF Pro 2超灵敏质谱仪,绘制青年(2月龄)、中年(12月龄)和老年(22月龄)小鼠的单个窦卵泡的蛋白质组图谱。通过差异表达蛋白质分析与蛋白质表达时间序列分析,寻找与年龄因素显著相关的蛋白质,解析窦卵泡的衰老图谱。结果本研究建立了一套针对1×10^(4)数量级细胞样本的蛋白质组学前处理流程,该流程仅需一步操作完成裂解和还原烷基化,酶切过程无需超声。即对于1×10^(4)数量级细胞样本,将裂解液中的脱氧胆酸钠浓度由1%提升至10%,可以使蛋白质平均鉴定种类从4089种增加到4389种,具有更佳的裂解效果(P<0.05);在裂解液中添加碳酸氢铵缓冲液(50 mmol·L^(-1),90μL),蛋白质平均鉴定种类从2579种显著提升至4389种(P<0.01);单胰蛋白酶与混合酶(Trypsin/Lys-C)具有相似的酶切效果,均能鉴定到约3950种蛋白质;将酶切时间由过夜缩短至0.5 h,蛋白质平均鉴定量由4299种提升至4632种(P<0.05),在节省时间的同时能获得更高的蛋白质鉴定量。与商业化试剂盒相比,本流程鉴定的蛋白质中约92.3%能被iST试剂盒或Ex极微量试剂盒鉴定,且在亲/疏水性、理论等电点�OBJECTIVE To establish a proteomic pre-processing protocol for low input samples(1×10^(4) cells)in order to map the proteomic aging atlas of individual mouse antral follicles and elucidate the aging patterns of mouse antral follicles.METHODS Using cell lines and primary mouse cells,the protocol was developed by optimizing lysis buffers,enzymatic digestion,and ultrasonication for 1×10^(4) cells(about 1μg protein).The sample pre-processing protocol established in this study was evaluated by comparing it with two commercial sample preparation kits:iST kit,EasyPept-Ex(Ex kit),in terms of protein identification overlap,overall grand average of hydropathy(GRAVY),theoretical isoelectric points,and protein molecular weight.The sample suitability of the current sample pretreatment process was assessed using primary spleen and liver cell samples of mice at three different scales:100,1000,and 10000.Using this sample pre-processing approach in conjunction with the highly sensitive timsTOF Pro 2 mass spectrometer,proteome maps of individual antral follicles from young-aged(2 months),middle-aged(12 months),and old-aged(22 months)mice were generated.Differential and time-series analyses identified age-related proteins,and elucidated the aging patterns of mouse antral follicles.RESULTS A proteomic pre-processing protocol for 1×10^(4) cell samples was established,requiring only one single-step operation for lysis,reduction,and alkylation,with enzymatic digestion not necessitating ultrasonication.We found that for cell samples on the order of 1×10^(4),increasing the concentration of sodium deoxycholate in the lysis buffer from 1%to 10%enhanced the number of identified proteins from 4089 to 4389,demonstrating improved lysis efficiency(P<0.05).The addition of ammonium bicarbonate buffer(50 mmol·L-1,90μL)to the lysis solution significantly increased the number of identified proteins from 2579 to 4389(P<0.01).Both single trypsin and mixed enzyme(trypsin/Lys-C)treatments yielded similar proteolytic outcomes,identifying approxima

关 键 词：蛋白质组学 样本前处理流程 卵巢衰老 窦卵泡 

分 类 号：R965[医药卫生—药理学]