Protein Stable Isotope Fingerprinting (P-SIF): A New Tool to Understand Natural Isotopic Heterogeneity of Mixed Microbial Ecosystems

Protein stable isotope fingerprinting (P-SIF) is a method to measure the carbon isotope ratios of whole proteins separated from complex mixtures, including cultures and environmental samples. The goal of P-SIF is to expose the links between identity and function in microbial ecosystems by (i) determining the ratios of 13C/12C (values of delta13C) for different taxonomic divisions, and (ii) using those values as clues to the metabolic pathways employed by the respective organisms, while (iii) not perturbing the system, ie, not adding exogenous substrates or isotope labels. To accomplish this, we employ two-dimensional HPLC to resolve a sample containing ca. 5-10 mg of mixed proteins into 960-1440 fractions. Each fraction then is split in two aliquots: The first is digested with trypsin for peptide sequencing, while the second is measured in triplicate using an isotope-ratio mass spectrometer interfaced with a spooling …