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Calculation of partial isotope incorporation into peptides measured by mass spectrometry.

Fetzer I, Jehmlich N, Vogt C, Richnow HH, Seifert J, Harms H, von Bergen M, Schmidt F - BMC Res Notes (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, D-04318 Leipzig, Germany. ingo.fetzer@ufz.de.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: To overcome the drawbacks of other calculation methods, we recently developed a new protein-based stable isotope probing (Protein-SIP) technique [6]. A brief overview of the workflow is displayed in Figure 1. In short, the incorporation of stable e.g.13C and/or15N isotopes from a substrate was used to pinpoint the metabolically active species within a consortium. Different species incorporate different degrees of the stable isotopes into their biomass according to their physiological ability and their activity (Figure 1A), which can be detected on peptide/protein level [7]. After cell harvesting and protein extraction, samples were analyzed by various proteomic and mass-spectrometry techniques. Due to the isotopic abundance of13C and/or15N, several isotopic envelopes can be detected by high-resolution mass spectrometry (MS) [8,9]. As expected, the isotopologues shifted to a higher mass range due to the incorporation of heavy labeled carbon into the proteins, as shown in the MS-spectrum in figure 1B. The12C-containing peptides are shown at the left hand side of each spectrum. A higher level of incorporation, as shown in the right spectra of part B, indicates a faster growth rate and/or an increased substrate usage of the carbon source of species. In order to assign these activities to different species, two steps have to be applied (i) the light peptides have to be identified by Tandem-MS (mass spectroscopy) and further assigned to the bacterial taxon and (ii) the heavy stable isotope incorporation has to be determined.


Calculation of partial isotope incorporation into peptides measured by mass spectrometry.

Fetzer I, Jehmlich N, Vogt C, Richnow HH, Seifert J, Harms H, von Bergen M, Schmidt F - BMC Res Notes (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913921&req=5

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.
Mentions: To overcome the drawbacks of other calculation methods, we recently developed a new protein-based stable isotope probing (Protein-SIP) technique [6]. A brief overview of the workflow is displayed in Figure 1. In short, the incorporation of stable e.g.13C and/or15N isotopes from a substrate was used to pinpoint the metabolically active species within a consortium. Different species incorporate different degrees of the stable isotopes into their biomass according to their physiological ability and their activity (Figure 1A), which can be detected on peptide/protein level [7]. After cell harvesting and protein extraction, samples were analyzed by various proteomic and mass-spectrometry techniques. Due to the isotopic abundance of13C and/or15N, several isotopic envelopes can be detected by high-resolution mass spectrometry (MS) [8,9]. As expected, the isotopologues shifted to a higher mass range due to the incorporation of heavy labeled carbon into the proteins, as shown in the MS-spectrum in figure 1B. The12C-containing peptides are shown at the left hand side of each spectrum. A higher level of incorporation, as shown in the right spectra of part B, indicates a faster growth rate and/or an increased substrate usage of the carbon source of species. In order to assign these activities to different species, two steps have to be applied (i) the light peptides have to be identified by Tandem-MS (mass spectroscopy) and further assigned to the bacterial taxon and (ii) the heavy stable isotope incorporation has to be determined.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, D-04318 Leipzig, Germany. ingo.fetzer@ufz.de.

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.

No MeSH data available.


Related in: MedlinePlus