摘要
【目的】利用稳定同位素代谢标记(stable-isotope labeling by amino acids in cell culture,SILAC)技术完成心肌细胞内蛋白的完整标记,为研究心肌缺氧等高原性心肌损伤提供相对定量的标准。【方法】分别采用中度和重度SILAC培养基培养心肌H9c2细胞,培养基中赖氨酸和精氨酸浓度分别为0.798和0.398 mmol/L;中度标记培养基含有赖氨酸4,4,5,5-D4-Lysine(Lys4)和精氨酸13C6-Arginine(Arg6);重度标记培养基含有赖氨酸13C615N2-Lysine(Lys8)和精氨酸13C615N4-Arginine(Arg10);通过连续培养6代后,提取细胞蛋白,采用过滤辅助蛋白组样本制备方法(Filter aided proteome preparation,FASP)联合12.5μg/ml质谱级胰蛋白酶溶液分别消化两种不同标记策略的细胞蛋白,经脱盐后进行高效液相结合质谱检测。所得数据由蛋白组学搜索软件MaxQuant进行分析,高斯拟合正态分布计算标记效率,GO分析鉴定蛋白参与的细胞生物学进程。【结果】中度SILAC标记组鉴定621个蛋白,正态分布μ值为5.06,标记效率为97.1%;重度SILAC标记组鉴定966个蛋白,正态分布μ值为5.98,标记效率为98.4%。这些蛋白主要集中于翻译、糖酵解等生物学进程。【结论】成功实现两种SILAC条件下心肌细胞H9c2蛋白的完整代谢标记,为研究高原性心肌损伤提供了蛋白组学的定量标准。
【Objective】To perform the complete labeling of proteins in rat cardiomyocytes via stable-isotope labeling by amino acids in cell culture(SILAC),and provide a relative quantitative standard for studying plateau myocardial injury.【Methods】Rat myocardial H9 c2 cells were cultured in medium-and heavy-labeled SILAC medium.The concentrations of arginine and lysine in the medium were0.398 and 0.798 mmol/L.The medium-labeled medium included 4,4,5,5-D4-Lysine(Lys4)and 13 C6-Arginine(Arg6),and the heavy-labeled medium included 13 C615 N2-Lysine(Lys8)and 13 C615 N4-Arginine(Arg10).After 6 generations of serial culture,proteins in medium-and heavy-labeled SILAC cells were extracted and digested with 12.5μg/ml trypsin by Filter aided proteome preparation(FASP).The digested peptides were detected by Ultra Performance Liquid Chromatography combined with Mass Spectrometry.The raw files of quantitative proteomics were analyzed by MaxQuant software,and labeling efficiency in medium-labeled and heavy-labeled SILAC was calculated according to the results of Gauss equation.The biological processes of identified proteins were analyzed by Gene Oncology(GO).【Results】There were 621 proteins qualified in the medium-labeling SILAC cells,and the labeling efficiency was 97.1%according toμ-value of normal distribution 5.06.There were 966 proteins qualified in the heavy-labeling SILAC cells,and the labeling efficiency was 98.4%according toμ-value of normal distribution 5.98.The identified proteins were mainly involved in many biological processes,such as translation and glycolysis.【Conclusion】The metabolic labeling is successfully completed in rat cardiomyocytes in medium-and heavy-labeled SILAC condition.It provides an excellent quantitative standard of proteomics for the study of plateau cardiomyopathy.
作者
张西南
张妍
徐忠伟
温晓昶
郭佳
邹爽
ZHANG Xi-nan;ZHANG Yan;XU Zhong-wei;WEN Xiao-chang;GUO Jia;ZOU Shuang(Department of Medical Service,Tibet Corps of PAP,Lasa 850003,China)
出处
《武警后勤学院学报（医学版）》
CAS
2020年第2期7-11,共5页
Journal of Logistics University of PAP（Medical Sciences）
基金
武警后勤学院基金项目(WHJ201906)
关键词
心肌细胞H9c2
定量蛋白质组学
稳定同位素代谢标记
生物进程分析
Cardiomyocytes H9c2
Quantitative proteomics
Stable-isotope labeling by amino acids in cell culture(SILAC)
Biological process
作者简介
张西南,本科,主治医师,主要从事高原缺氧研究工作;通信作者:邹爽,硕士,实验师,主要从事军事医学研究,E-mail:zoushuang98760@126.com。
