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

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.