Uncertainty analysis of isotopomer mass distribution: a quality control tool in metabolomics and fluxomics.

摘要

A combination of a bottom-up uncertainty study and MCM was for the first time applied to investigate the sources of variability that affect a typical MS-based fluxomics experiment. (1) The study shows that repeatability precision is the major uncertainty source. It is representative for the performance of the chromatographic separation (e.g. peak shape, retention time shift and integration procedures). Another relevant source of uncertainty are the ~(13)C and ~(12)C impurities present in the substrate but those are only affecting only the IF~(+0) and IF~(+1) respectively. Moreover the uncertainty of peak area increases with decreasing abundance of the different species, but is negligible compared to the other sources of uncertainty; (2) The above mentioned result is mainly highlighted by the data obtained from LC-MS/MS, which offers peaks with excellent signal to noise ratio, but where the scan rate can represent a significant bottleneck in case of too many or low abundant isotope-containing species in the mixture. GC-MS and LC-TOFMS are on the other hand instruments, which are more prone to mass interferences and demand for highly efficient chromatographic separations, especially in the case of complex biological matrices; (3) The MCM was successfully used to assess the overall uncertainty of the analytical platform determining IFs. MCM and GUM uncertainty always agreed within 1%.