Decimal Place Slope, A Fast and Precise Method for Quantifying 13C Incorporation Levels for Detecting the Metabolic Activity of

The metabolic incorporation of stable isotopes such as 13C or 15N into proteins has become a powerful tool for qualitative and quantitative proteome studies. We re-cently introduced a method that monitors heavy isotope incorporation into proteins and presented data revealing the metabolic activity of various species in a microbial consortium using this technique. To further develop our method using an liquid chromatography (LC)-mass spec-trometry (MS)-based approach, we present here a novel approach for calculating the incorporation level of 13C into peptides by using the information given in the decimal places of peptide masses obtained by modern high-re-solution MS. In the present study, the applicability of this approach is demonstrated using Pseudomonas putida ML2 proteins uniformly labeled via the consumption of [13C6]benzene present in the medium at concentrations of 0, 10, 25, 50, and 100 atom %. The incorporation of 13C was calculated on the basis of several labeled peptides derived from one band on an SDS-PAGE gel. The accu-racy of the calculated incorporation level depended upon the number of peptide masses included in the analysis, and it was observed that at least 100 peptide masses were required to reduce the deviation below 4 atom %. This accuracy was comparable with calculations of incorpo-ration based on the isotope envelope. Furthermore, this method can be extended to the calculation of the label-ing efficiency for a wide range of biomolecules, includ-ing RNA and DNA. The technique will therefore allow a highly accurate determination of the carbon flux in mi-crobial consortia with a direct approach based solely o