Comparison of extraction conditions and normalization approaches for cellular metabolomics of adherent growing cells with GC-MS

Common extraction protocols and sampling procedures for GC-MS metabolite profiling were applied to the MCF-7 breast cancer cell culture as a model system of adherent growing cells and validated for repeatability and reproducibility. For normalization of a concentration series after methanolic extraction, results obtained with cell count normalization equalled normalization to the chromatogram total ion current as a chromatogram-intrinsic parameter, indicating that cell counting as an additional experimental step could be omitted. However, we show here that both normalization strategies should only be applied for comparison of extracts with similar concentrations complicating comparisons of different samples, e. g. with different biological origin. Therefore, the application of TIC thresholds for the comparison of differently concentrated extracts is recommended with respect to the accuracy of the data, the working effort and complexity of the biological experiment. For proof of concept, MCF-7 cell samples generated by different sampling procedures were assessed using these thresholds. Within this context, direct extraction of adherent growing cells without any further preparation steps, which allows for rapid quenching of cell metabolism and efficient sample extraction, is discussed as an alternative to conventional cell counting and extraction. In conclusion, we recommend the consideration of chromatogram intensity thresholds for the mean comparison of differently concentrated sample replicates in GC-MS metabolite profiling.