基于平行反应监测验证技术分析绵羊胚胎骨骼肌蛋白质组数据

旨在对绵羊胚胎骨骼肌蛋白质组学数据进行分析，为阐明绵羊胚胎骨骼肌生长发育机制奠定基础。前期试验应用串联质谱定量法（tandem mass tag，TMT）对成年中国美利奴绵羊妊娠第85（D85）、105（D105）和135天（D135）的胚胎背最长肌进行蛋白质定量，并对D85/D105、D105/D135和D85/D135 3个比较组进行分析。本试验在此基础上，利用KEGG和平行反应监测（parallel reaction monitoring，PRM）等数据分析方法对3个比较组绵羊胚胎骨骼肌上、下调差异丰度蛋白质进行分析和验证，并对候选蛋白质进行生物信息学分析。本研究对肌纤维成熟分化标志性蛋白肌球蛋白重链（myosin-2 isoform X2，MYH）进行PRM靶向定量验证，结果表明，其变化趋势与绵羊胚胎肌纤维成熟分化趋势相符。本研究对上调差异丰度蛋白和下调差异丰度蛋白的KEGG分析结果表明，D105/D85比较组中上调差异丰度蛋白质显著富集于胰岛素等信号通路，D135/D105和D135/D85比较组中下调差异丰度蛋白质显著富集于DNA复制和蛋白质消化吸收等信号通路。与肌肉发育相关的cAMP依赖性蛋白激酶A催化亚基α（PRKACA）和葡萄糖转运蛋白成员4（GLUT4）蛋白均显著富集于胰岛素信号通路中，生物信息学分析显示，这两个蛋白质的理论分子量分别为121.73和20.64 ku，分别有147、12个带正电荷的氨基酸残基和135、12个带负电荷的氨基酸残基；理论等电点分别为8.81和6.54；亲水性平均系数分别为-0.408和0.811；PRKACA蛋白无N端糖基化位点，有45个磷酸化位点；GLUT4蛋白有1个N端糖基化位点，25个磷酸化位点；PRKACA蛋白三级结构与CAMP-2依赖蛋白激酶a嵌合融合的晶体结构相似度为85%，GLUT4蛋白三级结构与人葡萄糖转运蛋白GLUT1的相似性为78%。本研究表明，PRM定量验证趋势与绵羊胚胎肌纤维成熟分化趋势相符，PRKACA和GLUT4蛋白具有丰富的磷酸化修饰位点，并参与胰岛素信号通路调控，是重要的候选蛋白。

The Data Analysis of Embryonic Skeletal Muscle Proteomic in Sheep Based on Parallel Reaction Monitoring Technology

The purpose of this study was to analyze the data of embryonic skeletal muscle proteomic in sheep, and provide a basis for revealing the mechanism of embryonic skeletal muscle development and growth in sheep. In previous proteomic study, the embryonic longissimus dorsi proteins were quantified by using tandem mass tag (TMT) in the adult Chinese merino ewes on the Day 85 (D85), 105 (D105) and 135 (D135) of gestation, and 3 comparable groups D85/D105, D105/D135 and D85/D135 were set up. The KEGG and parallel reaction monitoring (PRM) methods were used to analyze and verify the up-and down-regulation differential abundance proteins in sheep embryonic skeletal muscle in 3 comparable groups, and bioinformatics analysis was performed for the candidate proteins. The marker protein about muscle fibers maturation and differentiation of myosin-2 isoform X2 (MYH) was verified by PRM, the results demonstrated that the change trend of MYH was consistent with the trend of sheep embryonic muscle fiber maturation and differentiation. The up-regulation and down-regulation differential abundance proteins were analyzed by KEGG method, the results showed that the up-regulation differen-tial abundance proteins in D105/D85 comparable group were significantly enriched in insulin signaling pathway, and the down-regulation differential abundance proteins in D135/D105 and D135/D85 comparable groups were significantly enriched in DNA replication and protein digestion and absorption signaling pathways. The muscle development related proteins, cAMP-dependent protein kinase catalytic subunit alpha (PRKACA) and glucose transporter member 4 isoform X1 (GLUT4), were significantly enriched in insulin signaling pathway. The bioinformatics analysis of the two proteins revealed that the theoretical molecular weight of PRKACA and GLUT4 were 121.73 and 20.64 ku, respectively. The PRKACA contained 147 positively charged amino acid residues and 135 negatively charged amino acid residues, the theoretical isoelectric point was 8.81, the hydrophilic average coefficient was -0.408; and the GLUT4 contained 12 positively charged amino acid residues and 12 negatively charged amino acid residues, the theoretical isoelectric point was 6.54, the hydrophilic average coefficient was 0.811. There were no N-terminal glycosylation site and there was 45 phosphorylation sites in PRKACA; there was an N-terminal glycosylation site and 25 phosphorylation sites in GLUT4. The homology of PRKACA and CAMP-2 relies on the chimeric fusion crystal structure of protein kinase a was 85% in tertiary structure, the homology of the GLUT4 and human glucose transporter GLUT1 was 78% in tertiary structure. The results showed that the trend of MYH quantification verified by PRM was consistent with the trend of embryonic muscle fiber maturation and differentiation. PRKACA and GLUT4 were important candidate proteins, with abundant phosphorylation sites, involved in the regulating of insulin signaling pathway.