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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

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.
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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.

Mentions: In order to determine the minimal number of needed peptide masses with an estimated incorporation accuracy of less than 5%, we additionally calculated an in silico peptide dataset with 50 atom % isotope incorporation of M. tuberculosis. We sub-sampled then the two already existing 0 and 100 atom, and the newly created 50%13C datasets by randomly drawing groups of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, and 1,000 theoretical peptides across all peptide lengths with no repetition for 100 replications. For each within-group sub-sample, we calculated the median and the standard error of each subgroup. A resulting box-and-whisker plot for each drawing shows the accuracy of the incorporation as a function of number of peptide masses (Figure 6).


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)

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

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

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.
Mentions: In order to determine the minimal number of needed peptide masses with an estimated incorporation accuracy of less than 5%, we additionally calculated an in silico peptide dataset with 50 atom % isotope incorporation of M. tuberculosis. We sub-sampled then the two already existing 0 and 100 atom, and the newly created 50%13C datasets by randomly drawing groups of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, and 1,000 theoretical peptides across all peptide lengths with no repetition for 100 replications. For each within-group sub-sample, we calculated the median and the standard error of each subgroup. A resulting box-and-whisker plot for each drawing shows the accuracy of the incorporation as a function of number of peptide masses (Figure 6).

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