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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.


Protein turnover demonstrated in mass spectra for a peptide from [13C]-labeled ATP synthase CF1 β-subunit. Arabidopsis plants were grown with 13C-carbon dioxide in the enclosure for three weeks from seed then transferred to ambient air for 0 h, 24 h, 48 h and 96 h before the leaves were harvested for total protein extraction. Unlabeled proteins were added as a 'spike' to the [13C]-labeled samples before gel electrophoresis, protein band isolation, in-gel trypsin digestion and LC-MS/MS analysis. The observed spectra were fitted with three β-binomial distributions: natural abundance (green); newly synthesized peptide (red); and old peptide (blue) distributions shown for each spectrum in the insets. Sample spectra of the tryptic peptide from ATP synthase CF1 β -subunit (FVQAGSEVSALLGR, C63H104N18O20) show the disappearance of the 13C-labeled peptide over time. This peptide was doubly charged with a monoisotopic m/z of 717.391. In addition to a shift in the fractional isotopic abundance newly synthesized peptide (red) with time, the distribution abundance ratios of the newly synthesized peptide (red) and old peptide populations (blue) increase with time.
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Figure 3: Protein turnover demonstrated in mass spectra for a peptide from [13C]-labeled ATP synthase CF1 β-subunit. Arabidopsis plants were grown with 13C-carbon dioxide in the enclosure for three weeks from seed then transferred to ambient air for 0 h, 24 h, 48 h and 96 h before the leaves were harvested for total protein extraction. Unlabeled proteins were added as a 'spike' to the [13C]-labeled samples before gel electrophoresis, protein band isolation, in-gel trypsin digestion and LC-MS/MS analysis. The observed spectra were fitted with three β-binomial distributions: natural abundance (green); newly synthesized peptide (red); and old peptide (blue) distributions shown for each spectrum in the insets. Sample spectra of the tryptic peptide from ATP synthase CF1 β -subunit (FVQAGSEVSALLGR, C63H104N18O20) show the disappearance of the 13C-labeled peptide over time. This peptide was doubly charged with a monoisotopic m/z of 717.391. In addition to a shift in the fractional isotopic abundance newly synthesized peptide (red) with time, the distribution abundance ratios of the newly synthesized peptide (red) and old peptide populations (blue) increase with time.

Mentions: As discussed above, an important goal of this research was to develop a high throughput method to measure protein turnover using whole plant stable isotope labeling via LC-MS/MS on a proteomic scale. A protein turnover measurement using this enclosure with 13CO2 was demonstrated as shown in Figure 3. To assist the identification of partially labeled peptides in the raw MS/MS using a standard search algorithm, unlabeled proteins were added to [13C]-labeled proteins in a 1:4 ratio (unlabeled to labeled) before being separated by SDS-PAGE. A predominant protein band around 52 kDa containing mostly Rubisco large subunit was excised and subjected to in-gel trypsinization prior to LC-MS/MS analysis. Several proteins including Rubisco large subunit and ATP synthase CF1 β-subunit were identified in these samples with multiple high confidence peptide assignments. We are interested in measuring the turnover of β-subunit of ATP synthase because we have previously observed this protein to be modified by the plant hormone indole 3-acetic acid in other plant species [36]. Once unlabeled peptides from this protein were identified, both unlabeled and labeled peptides were confirmed as coeluting sets of isotopic distributions by linear correlation of extracted isotopic channels within known retention time windows.


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)

Protein turnover demonstrated in mass spectra for a peptide from [13C]-labeled ATP synthase CF1 β-subunit. Arabidopsis plants were grown with 13C-carbon dioxide in the enclosure for three weeks from seed then transferred to ambient air for 0 h, 24 h, 48 h and 96 h before the leaves were harvested for total protein extraction. Unlabeled proteins were added as a 'spike' to the [13C]-labeled samples before gel electrophoresis, protein band isolation, in-gel trypsin digestion and LC-MS/MS analysis. The observed spectra were fitted with three β-binomial distributions: natural abundance (green); newly synthesized peptide (red); and old peptide (blue) distributions shown for each spectrum in the insets. Sample spectra of the tryptic peptide from ATP synthase CF1 β -subunit (FVQAGSEVSALLGR, C63H104N18O20) show the disappearance of the 13C-labeled peptide over time. This peptide was doubly charged with a monoisotopic m/z of 717.391. In addition to a shift in the fractional isotopic abundance newly synthesized peptide (red) with time, the distribution abundance ratios of the newly synthesized peptide (red) and old peptide populations (blue) increase with time.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Protein turnover demonstrated in mass spectra for a peptide from [13C]-labeled ATP synthase CF1 β-subunit. Arabidopsis plants were grown with 13C-carbon dioxide in the enclosure for three weeks from seed then transferred to ambient air for 0 h, 24 h, 48 h and 96 h before the leaves were harvested for total protein extraction. Unlabeled proteins were added as a 'spike' to the [13C]-labeled samples before gel electrophoresis, protein band isolation, in-gel trypsin digestion and LC-MS/MS analysis. The observed spectra were fitted with three β-binomial distributions: natural abundance (green); newly synthesized peptide (red); and old peptide (blue) distributions shown for each spectrum in the insets. Sample spectra of the tryptic peptide from ATP synthase CF1 β -subunit (FVQAGSEVSALLGR, C63H104N18O20) show the disappearance of the 13C-labeled peptide over time. This peptide was doubly charged with a monoisotopic m/z of 717.391. In addition to a shift in the fractional isotopic abundance newly synthesized peptide (red) with time, the distribution abundance ratios of the newly synthesized peptide (red) and old peptide populations (blue) increase with time.
Mentions: As discussed above, an important goal of this research was to develop a high throughput method to measure protein turnover using whole plant stable isotope labeling via LC-MS/MS on a proteomic scale. A protein turnover measurement using this enclosure with 13CO2 was demonstrated as shown in Figure 3. To assist the identification of partially labeled peptides in the raw MS/MS using a standard search algorithm, unlabeled proteins were added to [13C]-labeled proteins in a 1:4 ratio (unlabeled to labeled) before being separated by SDS-PAGE. A predominant protein band around 52 kDa containing mostly Rubisco large subunit was excised and subjected to in-gel trypsinization prior to LC-MS/MS analysis. Several proteins including Rubisco large subunit and ATP synthase CF1 β-subunit were identified in these samples with multiple high confidence peptide assignments. We are interested in measuring the turnover of β-subunit of ATP synthase because we have previously observed this protein to be modified by the plant hormone indole 3-acetic acid in other plant species [36]. Once unlabeled peptides from this protein were identified, both unlabeled and labeled peptides were confirmed as coeluting sets of isotopic distributions by linear correlation of extracted isotopic channels within known retention time windows.

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.