Quantification of the necessary labelling input in protein stable isotope probing

Abstract

Even though protein stable isotope probing is at least 100-times more sensitive than nucleic acid approaches, it is still unknown how much labelling input is necessary to not lose incorporation. Here, I used the average peptide model together with primary incorporation rates from previous experiments to determine the minimally necessary labelling input to detect incorporation of atoms present in proteins. Carbon-13 showed the highest efficiency for incorporation with detection misses only below 20.24 atom% labelling input. Despite the highest abundance in proteins, deuterium incorporation is not lost with at least 32.22 atom% labelling input due to instable acidic protons that undergo HD-exchange. All incorporation of oxygen-18 is detected with at least 28.15 atom% labelling input due to the +2 mass shift between its isotopes making up for its low abundance in proteins. Nitrogen-15 is less abundant in proteins and therefore harbors the most inefficient incorporation, resulting in a minimally necessary labelling input of 73.10 atom% labelling input. Altogether, these results can be used to properly design stable isotope probing experiments with the aim to maximize incorporation that can be extrapolated to other nucleic acid approaches due to similar incorporation routes.