Applying metabolic profiling techniques for stimulus-response experiments: chances and pitfalls.
So far it is mainly transcriptome and proteome analysis that has been applied to elucidate the correlation between genotype and phenotype although thorough metabolome studies can provide substantial information on the control of the metabolism at the biochemical level. Stimulus-response experiments, i.e. the investigation of metabolism dynamics after a glucose pulse (pulse experiment), can be used to study the in vivo enzyme kinetics offering insight into underlying reaction mechanisms. Usually, this requires rapid cell quenching combined with cell inactivation to'freeze' the microbial metabolism response at a definite time-lag after pulse stimulation. To access the 'frozen' metabolic reply, adequate analytical methods are needed to measure intracellular metabolite concentrations in the cell extract. As shown in the introductory review part, stimulus-response experiments were usually applied to study central metabolism dynamics in wildtype strains. Our own results, presented in the second part of the contribution, indicate that stimulus-response experiments should also be applied to analyse pathway dynamics in anabolic routes. Using the example of the aromatic amino acid pathway, an LC-MS/MS technique is presented that allows the quantification of intracellular pools of central metabolism as well as of the aromatic amino acid pathway. Based on the analytical approach metabolic profiling is performed to monitor the metabolism dynamics after a glucose pulse experiment allowing the conclusion that pulse stimulation is transmitted to the anabolic pathway of interest.
Oldiges
M
M
Institute of Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Takors
Ralf
R
D000465:Algorithms; D019149:Bioreactors / Q000382:microbiology*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D017022:Flow Injection Analysis / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008954:Models, Biological; D010649:Phenylalanine / Q000096:biosynthesis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000502:physiology; D013269:Stochastic Processes
D029968:Escherichia coli Proteins; D010649:Phenylalanine