Tracing incorporation of heavy water into proteins for species-specific metabolic activity in complex communities

Quantification of the necessary labelling input in protein stable isotope probing

“…Using the average model of an amino acid of C 4.94 H 7.76 N 1.14 O 1.48 S 0.04 (Senko, Beu and McLaffertycor, 1995), the number of incorporated isotopes for C, H, N and O of expected peptides ( n isotopes ) from the mass spectrometric measurements ranging from 8 to 32 amino acids ( n aa ) was estimated in steps of 5% with respect to the atom% of the input material ( RIA input ) and the primary incorporation rate a 1st (carbon-13 = 0.5 (Taubert et al ., 2012), deuterium = 0.2 (Taubert et al ., 2018; Starke et al ., 2020), nitrogen-15 = 0.6 (Starke, Kermer, et al ., 2016) and oxygen-18 = 0.6 (Starke et al ., 2020)) according to Equation 1. …”

“…The decomposition of nitrogen-15-labelled tobacco was followed in proteins of a soil microbial community (Starke, Kermer, et al ., 2016), sulfur-34 into amino acids (F.-A. Herbst et al ., 2013) as well as deuterium (Taubert et al ., 2018; Starke et al ., 2020) and oxygen-18 (Starke et al ., 2020) from heavy water as activity tracer in groundwater microbes. Otherwise, oxygen-17, sulfur-33 and sulfur-36 have not yet been utilized in protein-SIP, presumably due to the high cost of labelled material but also the low abundance of sulfur in biomolecules.…”

“…when degrading an entirely carbon-13-labelled hydrocarbon, carbon-13 from CO 2 could be incorporated to dilute the atom% of the input material (Taubert et al ., 2012; Starke, Keller, et al ., 2016), (ii) cells in natural systems are in different states of growth and will therefore incorporate isotopes at a differential efficiency and (iii) the incubation times are too short to achieve complete incorporation, or any combination of these. The incorporation rates as maximum of the Gaussian distribution compared to the labelling input as RIA value were previously determined to be 0.5 (or 50%) for carbon-13 (Taubert et al ., 2012), 0.2 (or 20%) for deuterium (Taubert et al ., 2018; Starke et al ., 2020), 0.6 (or 60%) for nitrogen-15 (Starke, Kermer, et al ., 2016) and 0.6 (or 60%) for oxygen-18 (Starke et al ., 2020). The relative difference between the monoisotopic peak at 0.00 (or 0%) and the maximum of the Gaussian distribution is explained as labelling ratio (LR).…”

“…Consistent with my hypothesis, carbon-13 showed the best efficiency of detecting incorporation when at least 20.24 atom% input label are supplied, resulting from the high number of carbon atoms in proteins ( Figure 2 ). With a higher abundance in proteins, deuterium showed less incorporation efficiency requiring at least 32.22 atom% of input material because of the low incorporation rate of only 20% (Taubert et al ., 2018; Starke et al ., 2020). This is because of the loss of acidic deuteria in the HD-exchange (Molday, Englander and Kallen, 1972; Bai et al ., 1993) once in contact with water during sample preparation and measurement.…”

Protein stable isotope probing with H218O differentiated cold stress response at permissive temperatures from general growth at optimal conditions in Escherichia coli K12

“…Another disadvantage is the necessity of adding an isotopically labeled substrate to the medium and thus bearing the risk of altering the community. A possible solution is the determination of the baseline metabolic activity with heavy water, either with D 2 O 11,12 or with H 2 18 O 11 . However, the species‐specific response to stress may alter which proteins are newly synthesized.…”