Fig. 3From: Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomesThe 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 S3Back to article page