Exploring the limits of robust detection of incorporation of 13C by mass spectrometry in protein-based stable isotope probing (protein-SIP)

One of the features of protein-based stable isotope probing is the parallel identification of differentially labeled peptide forms and the accurate calculation of their relative isotope abundances. The level of incorporation is informative of the metabolic activity of the species that synthesized the said protein and peptide. To model the carbon flux in a microbial community, an accurate assessment is crucial. Since the initial processes in carbon consumption are one of the most interesting objectives in microbial ecology, the methodology to detect low amounts of incorporation was tuned, and the limits of robust detection were analyzed. For this, Pseudomonas fluorescens DSM 50090T was grown on galactose using different ratios of C-12/C-13 galactose from 10% down to 0.1% labeled galactose. After prolonged cultivation to ensure complete labeling, protein samples were separated by one-dimensional gel electrophoresis, subsequently tryptically digested and analyzed by ultra-performance liquid chromatography (UPLC) Orbitrap tandem mass spectrometry (MS/MS) measurements. The isotopic patterns from identified peptides in the mass spectra were used to calculate the C-13 relative isotope abundance (RIA) in the respective peptides. The statistic distribution of the RIA values in dependence of the number of analyzed peptides was compared between the different ratios of unlabeled/labeled substrate. The acquired data showed that the applied method is capable of detecting a difference in C-13 incorporation of +/- 0.1% RIA based on at least 20 peptides. This sensitivity makes protein-stable isotope probing a valuable method for quantitative assessment of species specific metabolic activity in metaproteomics.