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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

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.
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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.

Mentions: During the run of the script, a window opens and the user has to indicate where the ASCII.txt file is located. The file should have the values of the peptide masses containing one peptide mass (z = 1 after deconvolution) per line with no header and/or row names. After selecting the file, the calculation starts immediately and results in a scatter-plot with lines displaying the 0 atom % and 100 atom % incorporation reference lines, the user's peptide masses, the fitting line of the data with the standard error as quality proxy, and the estimated incorporation including the standard error estimation (Figure 5). In a loop process, the estimation of multiple measurement campaigns can be carried out.


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)

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

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

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.
Mentions: During the run of the script, a window opens and the user has to indicate where the ASCII.txt file is located. The file should have the values of the peptide masses containing one peptide mass (z = 1 after deconvolution) per line with no header and/or row names. After selecting the file, the calculation starts immediately and results in a scatter-plot with lines displaying the 0 atom % and 100 atom % incorporation reference lines, the user's peptide masses, the fitting line of the data with the standard error as quality proxy, and the estimated incorporation including the standard error estimation (Figure 5). In a loop process, the estimation of multiple measurement campaigns can be carried out.

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