Limitations in detection of 15N incorporation by mass spectrometry in protein-based stable isotope probing (protein-SIP)

The method of protein-based stable isotope probing (protein-SIP) has previously been shown to allow the modeling of carbon fluxes in microbial communities, thus tackling one of the key questions in microbial ecology. The method allows the analysis of stable isotope distribution in peptides, revealing metabolic activities of the species present in an ecosystem. Besides carbon, an application of protein-SIP with nitrogen is of interest for resolving the nitrogen fluxes in microbial communities. Thus, the sensitivity and reliability of a protein-SIP approach employing ¹⁵N was analyzed. For this, cultivations of Pseudomonas fluorescens ATCC 17483 with different ratios of ¹⁴N/¹⁵N were performed, from 10 % down to 0.1 % ¹⁵N. After incubation leading to complete labeling of biomass, proteins were extracted and separated by one-dimensional gel electrophoresis, followed by tryptic digest and UPLC Orbitrap MS/MS analysis. ¹⁵N relative isotope abundance (RIA) was calculated based on isotopic patterns from identified peptides in mass spectra. Proteomics data have been deposited to ProteomeXchange with identifier PXD000127. The distribution of ¹⁵N RIA values among peptides was analyzed in samples with different ¹⁵N amount, and potential causes for variations within individual samples of either technical or biological origin were investigated. Using a number of 50 peptides, significant differences (p?≤?0.05) in ¹⁵N incorporation were found between samples of different ¹⁵N RIA down to 0.1 %. The study demonstrates that protein-SIP using ¹⁵N is sufficiently sensitive for quantitative investigation of microbial activity in nitrogen cycling processes.