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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

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).
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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).

Mentions: where PM Trans = transposed peptide masses, PM = peptide masses, and DR = decimal residuals producing a plot as given in Figure 3. The value of 1,800 within the formula (1) was iteratively estimated. Gradually increase of this value made the bands steeper until reaching a maximal vertical position at a value of about 1,800. A value greater than 1,800 made the bands tilt towards the opposite direction. The following classification was conducted using the Hartigan and Wong algorithm for k-means clustering [29] with three (0; 2,000; 4,000 Da) and four (0; 1,600; 3,200; 4,800 Da) pre-set clustering centers for the 'light' (no13C incorporation) and 'heavy' (complete13C substitution) dataset. For the clustering, pre-set center mass values do not have to be overly accurate since the precise numbers are automatically determined during the clustering procedure from actual group means [30]. Finally, the original peptide masses were taken and ranked according to cluster affiliation of the corresponding transformed mass values. In a next step, 0 Da was added to the peptide mass values belonging to the first cluster, 1 Da was added to the values of the second cluster and so on. Plotting these new mass values versus their decimal residuals resulted in the final straight plot (Figure 4).


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)

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

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

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).
Mentions: where PM Trans = transposed peptide masses, PM = peptide masses, and DR = decimal residuals producing a plot as given in Figure 3. The value of 1,800 within the formula (1) was iteratively estimated. Gradually increase of this value made the bands steeper until reaching a maximal vertical position at a value of about 1,800. A value greater than 1,800 made the bands tilt towards the opposite direction. The following classification was conducted using the Hartigan and Wong algorithm for k-means clustering [29] with three (0; 2,000; 4,000 Da) and four (0; 1,600; 3,200; 4,800 Da) pre-set clustering centers for the 'light' (no13C incorporation) and 'heavy' (complete13C substitution) dataset. For the clustering, pre-set center mass values do not have to be overly accurate since the precise numbers are automatically determined during the clustering procedure from actual group means [30]. Finally, the original peptide masses were taken and ranked according to cluster affiliation of the corresponding transformed mass values. In a next step, 0 Da was added to the peptide mass values belonging to the first cluster, 1 Da was added to the values of the second cluster and so on. Plotting these new mass values versus their decimal residuals resulted in the final straight plot (Figure 4).

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