(210h) Tandem Mass Spectrometry: The Next Frontier in 13C-Metabolic Flux Analysis

Tandem mass spectrometry is a new measurement technique for quantifying isotopic labeling for ¹³C-metabolic flux analysis (¹³C-MFA). Recently, we developed a general mathematical framework based on isotopomer simulations for incorporating tandem MS data into ¹³C-MFA (Choi et al., Metab Eng 2011). Here, we present further advancements we have made towards establishing next-generation tandem MS based flux analysis, specifically: 1) measuring complete positional isotopomer distribution of aspartate by tandem MS; and 2) developing an elementary metabolite units (EMU) framework for modeling tandem mass isotopomers.

First, we developed a simple and accurate method for determining the complete positional isotopomer distribution of aspartate carbon atoms by gas chromatography tandem mass spectrometry (GC-MS/MS). To achieve this, we screened tandem MS spectra of the TBDMS-derivative of aspartate for daughter fragments with the necessary carbon atom fragmentations to fully determine all sixteen isotopomers of aspartate. We selected five fragments that provided a redundant set of labeling measurements to quantify all isotopomers by least-squares regression. We demonstrated the validity of our approach using ¹³C-labeled aspartate standards and natural aspartate.

Next, we extended our modeling framework for tandem MS simulations. Here, we present for the first time our new framework based on EMUs, which are computationally more efficient to evaluate compared to previous methods based on isotopomers/cumomers. The new mathematical framework, which we called EMU², is based on two-dimensional representation of isotopic-labeling. Using this new formulation we take full advantage of the additional information that is obtained from tandem MS measurements for ¹³C-MFA.