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Abstract:
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 taxonomic identity and metabolic function in microbial ecosystems. To accomplish this, two dimensions of
chromatography are used in sequence to resolve a sample
containing ca. 5−10 mg of mixed proteins into 960 fractions.
Each fraction then is split in two aliquots: The
first is digested with trypsin for peptide sequencing, while the second has its ratio of 13C/12C (value of δ13
C) measured in triplicate using an isotope-ratio mass spectrometer interfaced with a spooling wire microcombustion device. Data from cultured species show that
bacteria have a narrow distribution of protein δ13
C values within individual taxa (±0.7−1.2%,1σ). This is moderately larger than the mean precision of the triplicate isotope measurements (±0.5‰,1σ) and may reflect heterogeneous distribution of 13C among the amino acids. When cells from different species are mixed together prior to protein extraction and separation, the results can predict accurately (to within ±1σ) the δ13C values of the original taxa. The number of data points required for this
endmember prediction is ≥20/taxon, yielding a theoretical resolution of ca. 10 taxonomic units/sample. Such resolution should be useful to determine the overall trophic breadth of mixed microbial ecosystems. Although we utilize P-SIF to measure natural isotope ratios, it also could be combined with experiments that incorporate stable isotope labeling.
