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Open Access Correspondence

Calculation of partial isotope incorporation into peptides measured by mass spectrometry

Ingo Fetzer1*, Nico Jehmlich4, Carsten Vogt3, Hans-Hermann Richnow3, Jana Seifert2, Hauke Harms1, Martin von Bergen2 and Frank Schmidt24

Author Affiliations

1 Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, D-04318 Leipzig, Germany

2 Helmholtz Centre for Environmental Research - UFZ, Department of Proteomics, Permoserstrasse 15, D-04318 Leipzig, Germany

3 Helmholtz Centre for Environmental Research - UFZ, Department of Isotope Biogeochemistry, Permoserstrasse 15, D-04318 Leipzig, Germany

4 Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Friedrich-Ludwig-Jahn-Strasse 15a, D-17487 Greifswald, Germany

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BMC Research Notes 2010, 3:178  doi:10.1186/1756-0500-3-178

Published: 24 June 2010

Abstract

Background

Stable isotope probing (SIP) technique was developed to link function, structure and activity of microbial cultures metabolizing carbon and nitrogen containing substrates to synthesize their biomass. Currently, available methods are restricted solely to the estimation of fully saturated heavy stable isotope incorporation and convenient methods with sufficient accuracy are still missing. However in order to track carbon fluxes in microbial communities new methods are required that allow the calculation of partial incorporation into biomolecules.

Results

In this study, we use the characteristics of the so-called 'half decimal place rule' (HDPR) in order to accurately calculate the partial13C incorporation in peptides from enzymatic digested proteins. Due to the clade-crossing universality of proteins within bacteria, any available high-resolution mass spectrometry generated dataset consisting of tryptically-digested peptides can be used as reference.

We used a freely available peptide mass dataset from Mycobacterium tuberculosis consisting of 315,579 entries. From this the error of estimated versus known heavy stable isotope incorporation from an increasing number of randomly drawn peptide sub-samples (100 times each; no repetition) was calculated. To acquire an estimated incorporation error of less than 5 atom %, about 100 peptide masses were needed. Finally, for testing the general applicability of our method, peptide masses of tryptically digested proteins from Pseudomonas putida ML2 grown on labeled substrate of various known concentrations were used and13C isotopic incorporation was successfully predicted. An easy-to-use script [1] was further developed to guide users through the calculation procedure for their own data series.

Conclusion

Our method is valuable for estimating13C incorporation into peptides/proteins accurately and with high sensitivity. Generally, our method holds promise for wider applications in qualitative and especially quantitative proteomics.