Calculation of partial isotope incorporation into peptides measured by mass spectrometry

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.

Figure 1

Schematic overview about the workflow of analysis. (A) Assimilation of heavy stable isotopes into the biomass of various species depends on the turnover and the interaction activity of the species. The incorporation of stable¹³C isotopes from a substrate can be used to pinpoint the metabolically active species within a consortium. The different incorporations are indicated by various amounts of label (gray-color scale). (B) After cell harvesting and protein extraction, samples were tryptically digested and analyzed by MS. The isotopologues shifted to a higher mass range due to their incorporation of heavy labeled carbon into the proteins. A higher level of incorporation indicates a faster growth rate and/or a primary role in the degradation of the labeled substrate within the food web. (C) The incorporation of heavy stable isotopes into peptides/proteins can be estimated using the HDPR. (D) Peptides can be used to obtain phylogenetic information (in case of unique peptides), for structural analysis, and for physiological information about the actual state of the microbial cells. (E) Based on this information, C-fluxes and food web structures can be elucidated and may further help to reconstruct the interaction of microbial communities.

Figure 2

A scatter plot of masses from 90,637 peptides (m/z) and decimal residuals (digits behind the decimal point) of Mycobacterium tuberculosis within a mass range of m/z 0-5,000. (A) Unlabeled sequences (0 atomic %¹³C incorporation), (B) fully labeled (100 atomic %¹³C incorporation). Lines depict the overlap of scatter groups.

Figure 3

A scatter plot of temporally transposed masses from 90,637 peptides (m/z) and decimal residuals of a Mycobacterium tuberculosis dataset (details see text)). Grey dots indicate cluster affiliation after classification by k-means clustering for (A) Unlabeled sequences (0 atomic %¹³C incorporation), (B) fully labeled (100 atomic %¹³C incorporation).

Figure 4

A scatter plot of 90,637 peptide masses (m/z) and their decimal residuals of a Mycobacterium tuberculosis dataset after classification and data modification for unlabeled (0 atom %¹³C incorporation; light grey) and fully labeled (100 atom %¹³C incorporation; dark grey) peptide masses resulted. The two lines represent the slopes for the calculation of¹³C incorporation. The values of the two scatters are for the unlabeled b_0% = 5.1357e-4 and b_100% = 6.3347e-4 for fully labeled dataset. Numbers in each segment represent Daltons added to decimal residuals to achieve straight plot from figure 3 (for details see text).

Figure 5

Typical plot resulting from user experimental measurements (dark circles), reference slope lines for unlabeled (0 atom %¹³C incorporation; dark grey line) and fully labeled (100 atom %¹³C incorporation; light grey line) and the slope calculated from user data measurements (thick black line). The¹³C incorporation for user data is directly calculated and displayed in the plot. This example consists of 150 practical peptides from Pseudomonas putida grown on fully labeled benzene.

Figure 6

Box-and-whisker plot for approximate accuracy of¹³C incorporation for groups of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, and 1000 peptides from 100 random draws (no repetition) for Mycobacterium tuberculosis peptide dataset of 0, 50 and 100 atom %¹³C incorporation. Boxes represent lower and upper quartile, whiskers the upper and lower (95%) confidence interval and thick lines in boxes the median within the sub-sample groups. The two thin black lines depict 5% incorporation limits and the thick black line depicts the number of samples necessary to cross the 5% incorporation limit.