Xing-Csrp3-protein measurments

To elucidate the mechanism by which CSRP3 modulates pig satellite cell differentiation, quantitative proteomics analysis of CSRP3-knockdown versus control pig satellite cells at day 2 of differentiation was performed. Proteins from pig satellite cells were extracted using RIPA buffer supplemented with PMSF and a protease inhibitor cocktail. Cell proteins were solubilized in 7M urea and 2M thiourea, and protein concentrations were quantified using the Bradford Protein Assay Kit. Proteins were digested following the FASP method. The resulting peptides were analyzed using a Q-Exactive high-resolution mass spectrometer coupled with a Nano-Acquity nano HPLC system. Data processing for peak picking and alignment was performed using Pyogenesis QI for Proteomics software (build 2.0, Nonlinear Dynamics, Newcastle, UK).

为阐明CSRP3调控猪卫星细胞分化的分子机制，本研究对分化第2天的CSRP3敲低（CSRP3-knockdown）组与对照组猪卫星细胞开展了定量蛋白质组学分析。采用添加了苯甲基磺酰氟（PMSF）与蛋白酶抑制剂混合物的RIPA缓冲液提取猪卫星细胞总蛋白；将细胞蛋白溶解于7M尿素与2M硫脲混合溶液中，并采用Bradford蛋白定量试剂盒测定蛋白浓度；采用FASP（Filter-aided sample preparation）法进行蛋白质酶解；所得肽段采用搭载Nano-Acquity纳升级高效液相色谱系统的Q-Exactive高分辨质谱仪进行分析；采用Pyogenesis QI for Proteomics软件（版本2.0，英国纽卡斯尔Nonlinear Dynamics公司开发）完成峰拾取与峰对齐的数据处理工作。