Protein Stable Isotope Fingerprinting: Multidimensional Protein Chromatography Coupled to Stable Isotope-Ratio Mass Spectrometry
Protein Stable Isotope Fingerprinting: Multidimensional Protein Chromatography Coupled to Stable Isotope-Ratio Mass Spectrometry
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DOI：
10.1021/ac502494b
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发表时间：
2014-09-02
影响因子：
7.4
通讯作者：
Pearson, Ann
中科院分区：
文献类型：
--
作者：
Mohr, Wiebke;Tang, Tiantian;Pearson, Ann
Protein stable isotope fingerprinting (P-SIP) 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 C-13/C-12 (value of delta C-13) 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 (delta C-13 values within individual taxa (+/- 0.7-1.2 parts per thousand, l sigma). This is moderately larger than the mean precision of the triplicate isotope measurements (+/- 0.5 parts per thousand, 1 sigma) and may reflect heterogeneous distribution of C-13 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 +/- 1 sigma) the delta C-13 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.
