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Fig. 3 | Microbiome

Fig. 3

From: Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes

Fig. 3

The number of labeled atoms per substrate molecule impacts the ability to quantify label incorporation accurately. Labeling, to saturation, of E. coli and B. subtilis with single-labeled (13C2) and fully labeled (13C1-6) glucose. The 13C/12C ratio in the substrate was varied. Note that unlabeled glucose (0% added 13C glucose) has a natural 13C content of around 1.1%. Each orange circle is the median 13C/12C ratio of all peptides measured in one replicate incubation (on average 2758 peptides per replicate). Determined 13C/12C ratios increased linearly with substate 13C/12C ratios (R2 > 0.999). Almost 100% of the substrate 13C was recovered in protein for 13C2 glucose labeled cells. Recovery was lower for 13C1-6 glucose. The proportion of neutron masses detected via the improved peptide identification strategy using N- and C-terminal modifications (yellow circles) increased with substrate 13C/12C ratios, but at low linearity and sensitivity. The number of Calis-p filtered peptide spectrum matches (PSM) decreased for 13C/12C ratios above 2.5% (insets) as expected based on Fig. 2 and Fig. 2. Assimilation of carbon into amino acids in clumps of multiple 13C atoms was detectable in peptide spectra of cultures fed with 13C1-6 glucose as shown in pie charts for experiments fed with 13C/.12C 1% above natural background. The detailed data for this figure can be found in Supplementary Table S3

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