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Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants
Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746739/ https://www.ncbi.nlm.nih.gov/pubmed/29137184 http://dx.doi.org/10.3390/metabo7040059 |
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author | Rossi, Merja T. Kalde, Monika Srisakvarakul, Chaiyakorn Kruger, Nicholas J. Ratcliffe, R. George |
author_facet | Rossi, Merja T. Kalde, Monika Srisakvarakul, Chaiyakorn Kruger, Nicholas J. Ratcliffe, R. George |
author_sort | Rossi, Merja T. |
collection | PubMed |
description | Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms suggest that the metabolic phenotype will differ between cell types. In silico analysis of simulated metabolite isotopomer datasets shows that cellular heterogeneity confounds conventional MFA because labelling data averaged over multiple cell types does not necessarily yield averaged flux values. A potential solution to this problem—the use of cell-type specific reporter proteins as a source of cell-type specific labelling data—is proposed and the practicality of implementing this strategy in the roots of Arabidopsis thaliana seedlings is explored. A protocol for the immunopurification of ectopically expressed green fluorescent protein (GFP) from Arabidopsis thaliana seedlings using a GFP-binding nanobody is developed, and through GC-MS analysis of protein hydrolysates it is established that constitutively expressed GFP reports accurately on the labelling of total protein in root tissues. It is also demonstrated that the constitutive expression of GFP does not perturb metabolism. The principal obstacle to the implementation of the method in tissues with cell-type specific GFP expression is the sensitivity of the GC-MS system. |
format | Online Article Text |
id | pubmed-5746739 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-57467392018-01-03 Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants Rossi, Merja T. Kalde, Monika Srisakvarakul, Chaiyakorn Kruger, Nicholas J. Ratcliffe, R. George Metabolites Article Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms suggest that the metabolic phenotype will differ between cell types. In silico analysis of simulated metabolite isotopomer datasets shows that cellular heterogeneity confounds conventional MFA because labelling data averaged over multiple cell types does not necessarily yield averaged flux values. A potential solution to this problem—the use of cell-type specific reporter proteins as a source of cell-type specific labelling data—is proposed and the practicality of implementing this strategy in the roots of Arabidopsis thaliana seedlings is explored. A protocol for the immunopurification of ectopically expressed green fluorescent protein (GFP) from Arabidopsis thaliana seedlings using a GFP-binding nanobody is developed, and through GC-MS analysis of protein hydrolysates it is established that constitutively expressed GFP reports accurately on the labelling of total protein in root tissues. It is also demonstrated that the constitutive expression of GFP does not perturb metabolism. The principal obstacle to the implementation of the method in tissues with cell-type specific GFP expression is the sensitivity of the GC-MS system. MDPI 2017-11-13 /pmc/articles/PMC5746739/ /pubmed/29137184 http://dx.doi.org/10.3390/metabo7040059 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Rossi, Merja T. Kalde, Monika Srisakvarakul, Chaiyakorn Kruger, Nicholas J. Ratcliffe, R. George Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants |
title | Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants |
title_full | Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants |
title_fullStr | Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants |
title_full_unstemmed | Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants |
title_short | Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants |
title_sort | cell-type specific metabolic flux analysis: a challenge for metabolic phenotyping and a potential solution in plants |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746739/ https://www.ncbi.nlm.nih.gov/pubmed/29137184 http://dx.doi.org/10.3390/metabo7040059 |
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