Decimal place slope, a fast and precise method for quantifying 13C incorporation levels for detecting the metabolic activity of microbial species

Abstract

The metabolic incorporation of stable isotopes such as (13)C or (15)N into proteins has become a powerful tool for qualitative and quantitative proteome studies. We recently 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 spectrometry (MS)-based approach, we present here a novel approach for calculating the incorporation level of (13)C into peptides by using the information given in the decimal places of peptide masses obtained by modern high-resolution MS. In the present study, the applicability of this approach is demonstrated using Pseudomonas putida ML2 proteins uniformly labeled via the consumption of [(13)C(6)]benzene present in the medium at concentrations of 0, 10, 25, 50, and 100 atom %. The incorporation of (13)C was calculated on the basis of several labeled peptides derived from one band on an SDS-PAGE gel. The accuracy 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 incorporation based on the isotope envelope. Furthermore, this method can be extended to the calculation of the labeling efficiency for a wide range of biomolecules, including RNA and DNA. The technique will therefore allow a highly accurate determination of the carbon flux in microbial consortia with a direct approach based solely on LC-MS.

Fig. 1.

The experimental design for determining the ¹³C incorporation level in peptides by its decimal place slope. In our study, P. putida ML2 was grown in the presence of [¹³C₆]benzene (sole energy and carbon source) and, as a control, in the presence of [¹²C₆]benzene. Subsequently, the proteins were extracted and subjected to 1-D gel electrophoresis and tryptic in-gel digestion, and the peptides were analyzed by nano-LC LTQ Orbitrap-MS. The difference in the isotopic pattern between the ¹²C/¹³C peptides allowed for the calculation of ¹³C incorporation levels for various labeled contents, using the highest intensity ¹³C peak and the decimal place slope script.

Fig. 2.

Averaged MS-spectra of one peptide from the benzene-1,2 dioxygenase ferredoxin-NAD(+) reductase subunit protein (P. putida KT2440) measured by nano-LC LTQ Orbitrap-MS to demonstrate the differential isotopic pattern of ¹²C peptides and ¹³C-labeled peptides during the incorporation of different substrate ratios. The measured peptide with the sequence GIFAVGDVATWPLHSGGK has a mass charge of +2 and is also listed in supplemental Table 1. At all substrate ratios, the isotopomers changed from the natural monoisotopic mass of 906.476 (A) because of the incorporation of heavy isotopes. The highest ¹³C peak in the 10 atom % labeling experiment (B) with [MH+H]⁺² was 909.48621. The highest ¹³C peak in the 25 atom % labeling experiment (C) with [MH+H]⁺² was 915.00487. C, The highest ¹³C peak in the 25 atom % labeling experiment was [MH+H]⁺² 915.00487. D, the highest ¹³C peak in the 50 atom % labeling experiment was [MH+H]⁺² 927.04526. E, the highest ¹³C peak in the 100 atom % ¹³C labeling experiment was [MH+H]⁺² 947.61397.

Fig. 3.

Practical data set of P. putida ML2 peptides measured by nano-LC LTQ Orbitrap-MS. In each case, exactly 100 peptide masses (highest intensity peak) from 22 different proteins were plotted according to their exact mass and decimal place. The slope of the regression line passing through the data points was calculated by means of an R-script, resulting in the given figures (A–E) for the different substrate ratios (0, 10, 25, 50, and 100 atom %). For example, the experimental incorporation level of 7.6 atom % (B) was calculated based upon the ¹²C (0.000512) and ¹³C slope (0.000632) for the 10 atom % labeling experiment.