定量监控大肠杆菌主代谢目标蛋白质及中间代谢物

Quantitative monitoring targeted proteins and intermediate metabolites in Escherichia coli primary metabolic pathways

摘要:

摘要:【目的】监控大肠杆菌(Escherichia coli)主代谢通路上蛋白表达状况及中间代谢物变化，为代谢工程改造提供基础性数据及检测方法。【方法】利用Skyline软件靶向设计主代谢(糖酵解途径、磷酸戊糖途径、三羧酸循环、混合酸发酵途径及脂肪酸合成途径)目标蛋白质label-free (MRM)方法对其相对定量监控;在相同质谱平台(Triple Quad 4500)上利用LC-MS/MS(MRM)方法对目标中间代谢物绝对定量监控。【结果】实验表明不同生长时期内(对数生长期、稳定期及衰亡期)大肠杆菌主代谢蛋白质表达表现出4种不同的变化现象，某一代谢通路上的单一蛋白不能反映该通路的表达状态;磷酸戊糖途径、混合酸发酵途径以及三羧酸循环途径中较多的蛋白质在衰亡期表达量最高，但几种目标中间代谢产物(ATP、ADP、AMP、NAD+、NADH、NADP+、NADPH、CoA、acetyl-CoA)的积累量与对数生长期相比，稳定期及衰亡期都相应减少(除了acetyl-CoA以外)。【结论】该文中使用的检测方法可以有效地反映大肠杆菌体内代谢的基本状况。

Abstract:

Abstract:［Objective］ This study aimed to monitor targeted protein expression levels and changes in intermediate metabolites in the primary metabolic pathways of Escherichia coli to obtain basic data and to develop testing methods that can be used in metabolic engineering．［Methods］We used the Skyline software to design a label-free method (multiple reaction monitoring) for relative quantitative monitoring of proteins from primary metabolic pathways (i.e.，glycolytic pathway，pentose phosphate pathway，mixed acid fermentation，tricarboxylic acid cycle，and fatty acid synthesis pathway)．We used the same mass spectrometry platform (Triple Quad 4500) for liquid chromatography-tandem mass spectrometry (multiple reaction monitoring) analysis of targeted intermediate metabolites during absolute quantitative monitoring.［Results］Protein expression in the primary metabolic pathways of E．coli showed four different phenomena in the different growth periods (exponential phase，stationary phase，and decline phase)． Expression levels of a single protein cannot provide accurate information regarding the status of these pathways． More proteins in the pentose phosphate pathway，mixed acid fermentation，and the tricarboxylic acid cycle showed the highest expression in the decline phase，but accumulation of several targeted intermediate metabolites (ATP，ADP，AMP，NAD+，NADH，NADP +，NADPH， CoA，and acetyl-CoA) in the stationary phase and decline phase correspondingly decreased compared to the levels in the exponential phase (in addition to acetyl-CoA).［Conclusion］ The detection methods used in this study can help determine the basic conditions of E．coli metabolism in vivo.