Limits...
An automated growth enclosure for metabolic labeling of Arabidopsis thaliana with 13C-carbon dioxide - an in vivo labeling system for proteomics and metabolomics research.

Chen WP, Yang XY, Harms GL, Gray WM, Hegeman AD, Cohen JD - Proteome Sci (2011)

Bottom Line: Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides.The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS.Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticultural Science, University of Minnesota, Saint Paul, USA. hegem007@umn.edu.

ABSTRACT

Background: Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with 13C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate 13C from 13CO2 gas. Because of the high cost of 13CO2 it is critical that the design conserve the labeled gas.

Results: A fully enclosed automated plant growth enclosure has been designed and assembled where the system simultaneously monitors humidity, temperature, pressure and 13CO2 concentration with continuous adjustment of humidity, pressure and 13CO2 levels controlled by a computer running LabView software. The enclosure is mounted on a movable cart for mobility among growth environments. Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides.

Conclusion: The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS. Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs.

No MeSH data available.


Related in: MedlinePlus

Amino acid enrichment and turnover in the leaves of Arabidopsis plants labeled with 13CO2. Arabidopsis seedlings were grown in the enclosure with 99 atom% 13CO2 for three weeks from seed then chased with ambient air (containing 12CO2) for different durations before sampling for amino acid analyses using GC-MS. Data from 3 amino acids, cysteine, asparagine and arginine, were not shown. The half-lives of amino acids were calculated by non-linear regression assuming a simple exponential decay process with no plateau. The percent 13C enrichment is given as the number listed parenthetically above the bars for each amino acid. A graph inside this figure shows the diminution of a heavy fragment ion (m/z 163) and emergence of the light fragment ion (m/z 160) of the derivatized [13C]-labeled aspartic acid (Asp) during the isotopic dilution period. The elemental composition of this specific fragment ion was determined to be C6H10NO4; but in the fragment only C3H4N1 originated from the amino acid and the remainder of the elements were from the derivatization reagent.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3046907&req=5

Figure 2: Amino acid enrichment and turnover in the leaves of Arabidopsis plants labeled with 13CO2. Arabidopsis seedlings were grown in the enclosure with 99 atom% 13CO2 for three weeks from seed then chased with ambient air (containing 12CO2) for different durations before sampling for amino acid analyses using GC-MS. Data from 3 amino acids, cysteine, asparagine and arginine, were not shown. The half-lives of amino acids were calculated by non-linear regression assuming a simple exponential decay process with no plateau. The percent 13C enrichment is given as the number listed parenthetically above the bars for each amino acid. A graph inside this figure shows the diminution of a heavy fragment ion (m/z 163) and emergence of the light fragment ion (m/z 160) of the derivatized [13C]-labeled aspartic acid (Asp) during the isotopic dilution period. The elemental composition of this specific fragment ion was determined to be C6H10NO4; but in the fragment only C3H4N1 originated from the amino acid and the remainder of the elements were from the derivatization reagent.

Mentions: In order to measure amino acid turnover, after an initial 13CO2 labeling period beginning at germination, [13C]-enrichment was monitored immediately after removal from the 13CO2 enclosure and at various periods of growth in ambient air (with 12CO2) to allow dilution of the isotope pools. Samples of [13C]-labeled Arabidopsis leaves were harvested at least 4 h after the start of the light period to reduce major metabolic changes due to the diurnal cycle [19]. No significant differences in plant morphology were observable throughout the three-week growth period between plants grown inside and outside the [13C]-enclosure (using otherwise identical conditions, in the same walk-in growth chamber). Of 17 amino acids monitored, 9 amino acids, including serine, glycine, alanine, methionine, glutamine, histidine, tyrosine, phenylalanine and tryptophan, were essentially fully enriched (> 98%) after 13CO2 labeling from the time of seed germination (Figure 2). The remaining observable amino acids showed enrichments of >93%, except for proline, which had only 85% enrichment.


An automated growth enclosure for metabolic labeling of Arabidopsis thaliana with 13C-carbon dioxide - an in vivo labeling system for proteomics and metabolomics research.

Chen WP, Yang XY, Harms GL, Gray WM, Hegeman AD, Cohen JD - Proteome Sci (2011)

Amino acid enrichment and turnover in the leaves of Arabidopsis plants labeled with 13CO2. Arabidopsis seedlings were grown in the enclosure with 99 atom% 13CO2 for three weeks from seed then chased with ambient air (containing 12CO2) for different durations before sampling for amino acid analyses using GC-MS. Data from 3 amino acids, cysteine, asparagine and arginine, were not shown. The half-lives of amino acids were calculated by non-linear regression assuming a simple exponential decay process with no plateau. The percent 13C enrichment is given as the number listed parenthetically above the bars for each amino acid. A graph inside this figure shows the diminution of a heavy fragment ion (m/z 163) and emergence of the light fragment ion (m/z 160) of the derivatized [13C]-labeled aspartic acid (Asp) during the isotopic dilution period. The elemental composition of this specific fragment ion was determined to be C6H10NO4; but in the fragment only C3H4N1 originated from the amino acid and the remainder of the elements were from the derivatization reagent.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046907&req=5

Figure 2: Amino acid enrichment and turnover in the leaves of Arabidopsis plants labeled with 13CO2. Arabidopsis seedlings were grown in the enclosure with 99 atom% 13CO2 for three weeks from seed then chased with ambient air (containing 12CO2) for different durations before sampling for amino acid analyses using GC-MS. Data from 3 amino acids, cysteine, asparagine and arginine, were not shown. The half-lives of amino acids were calculated by non-linear regression assuming a simple exponential decay process with no plateau. The percent 13C enrichment is given as the number listed parenthetically above the bars for each amino acid. A graph inside this figure shows the diminution of a heavy fragment ion (m/z 163) and emergence of the light fragment ion (m/z 160) of the derivatized [13C]-labeled aspartic acid (Asp) during the isotopic dilution period. The elemental composition of this specific fragment ion was determined to be C6H10NO4; but in the fragment only C3H4N1 originated from the amino acid and the remainder of the elements were from the derivatization reagent.
Mentions: In order to measure amino acid turnover, after an initial 13CO2 labeling period beginning at germination, [13C]-enrichment was monitored immediately after removal from the 13CO2 enclosure and at various periods of growth in ambient air (with 12CO2) to allow dilution of the isotope pools. Samples of [13C]-labeled Arabidopsis leaves were harvested at least 4 h after the start of the light period to reduce major metabolic changes due to the diurnal cycle [19]. No significant differences in plant morphology were observable throughout the three-week growth period between plants grown inside and outside the [13C]-enclosure (using otherwise identical conditions, in the same walk-in growth chamber). Of 17 amino acids monitored, 9 amino acids, including serine, glycine, alanine, methionine, glutamine, histidine, tyrosine, phenylalanine and tryptophan, were essentially fully enriched (> 98%) after 13CO2 labeling from the time of seed germination (Figure 2). The remaining observable amino acids showed enrichments of >93%, except for proline, which had only 85% enrichment.

Bottom Line: Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides.The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS.Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticultural Science, University of Minnesota, Saint Paul, USA. hegem007@umn.edu.

ABSTRACT

Background: Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with 13C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate 13C from 13CO2 gas. Because of the high cost of 13CO2 it is critical that the design conserve the labeled gas.

Results: A fully enclosed automated plant growth enclosure has been designed and assembled where the system simultaneously monitors humidity, temperature, pressure and 13CO2 concentration with continuous adjustment of humidity, pressure and 13CO2 levels controlled by a computer running LabView software. The enclosure is mounted on a movable cart for mobility among growth environments. Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides.

Conclusion: The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS. Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs.

No MeSH data available.


Related in: MedlinePlus