Intracellular Metabolome Analysis

On the other hand, Sellick et al. [96-98] studied the applicability of cold-methanol quenching for GS-CHO cells, finally concluding that CHO quenching in -40 °C cold 60% methanol supplemented with 0.85% (w/v) ammonium bicarbonate (pH 7.4) resulted in representative metabolite patterns. 

Ahn and Antoniewicz [119], and Niklas and Heinzle [120]. Because they already cover a large field of MFA appHcations, only some few items are highlighted in the following. 

One particularity of MFA in mammalian cells is its compartmentalization, making it necessary to consider exchange fluxes via shuttle systems for instance between mitochondria and cytoplasm. Obviously, the consideration of an additional compartment increases the model complexity, which may result in an underdetermined system. To enable identifiability of the model, one may simplify the same by ignoring cellular compartments, locating all reactions in a pseudo-prokaryotic manner. However, what is the impact on metabolic flux patterns  

The noncompartmented model consisted of 73 transformers (reaction rates and transport step), balancing 53 metaboHtes. The compartmented approach was buUt on 95 transformers and 75 metabolites. While most of the simulated fluxes were similar in both approaches, a fraction of approximately 15% of totally available ATP was missing in the compartmented model compared to the noncompartmented approach. This was due to the assumed activity of the citrate-pyruvate shuttle that imports pyruvate (via pyruvate/H+ symporter) into mitochondria by exporting citrate (via citrate/malate antiporter). In cytoplasm, citrate is further 

---

Raamsdonk and colleagues set out to develop a metabolomics method that could be used to characterize proteins of unknown function in yeast [14]. Using an NMR approach they analyzed intracellular metabolites in mutants of Saccharomyces cerevisiae. The resulting NMR spectra were then analyzed by multivariate analysis, including principle component analysis (PCA) to identify differences in the spectra that can distinguish different mutants (Fig. 2). Two important results came out of these studies that would reveal the value of metabolomics in biological research. 

A further advantage of combining IPC and MS is that isotope-labeled internal standards may be used for accurate quantification of analytes. No recovery check is needed for analytes because the labeled analogues of the analytes that serve as internal standards are added to the samples before the extraction step. This allows sample mixtures to be analyzed without pretreatment because the labeled internal standards share the fate of the un labeled analyte during sample processing without differences in extraction efficiency between the labeled standards and unlabeled analytes. This method achieved excellent precision and improved linearity of calibration lines despite interference from sample matrices in the quantitative analysis of important secondary intracellular metabolites in a complex biological sample solution from cultured cells. The technique is a valuable strategy for metabolomics [75],... 

Tlie analysis of the intracellular metabolites, the metabolome, by means of comparison of mutants metabolic profiles (comparative metabolomics) aids in understanding the networks of proteins and the presence of silent genes. 

---