Protocol for Performing Protein Stable Isotope Probing (Protein-SIP) Experiments

Abstract

Protein-based stable isotope probing (protein-SIP) was developed to link microbial-specific metabolic function to phylogenetic information. The SIP-concept is based on the application of stable isotope-labeled compounds, 13C or 15N as substrates supplied to microbial communities grown within microcosm systems. Microorganisms capable of utilizing these substrates become labeled upon metabolism and biosynthesis. The sum of all proteins represents the functional status of the living organisms, so that the degree of heavy isotope incorporation represents a proxy for substrate assimilation. The incorporation into peptides/proteins is calculated using the non-negative least squares approach. In addition, the peptide sequence can discern the active microorganisms resolved at species-level, if species specific genomic information and unique peptide information is available. The protein-SIP approach is a useful method to investigate the composition and the functional state of microbial communities. Here, we provide a guideline for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides the reader through the proteomics pipeline, including protein extraction, protein separation, and the mass spectrometric analysis. The calculation of stable isotope incorporation into peptides/proteins will be explained in more detail.