Limits...
Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

Jäpelt KB, Christensen JH, Villas-Bôas SG - Microb. Cell Fact. (2015)

Bottom Line: However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging.The implementation of a reliable, reproducible quenching method is essential within the metabolomics community.Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

View Article: PubMed Central - PubMed

Affiliation: Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. dkkba@chr-hansen.com.

ABSTRACT

Background: Quenching in cold buffered methanol at -40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline.

Results: Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in Lactobacillus paracasei subsp. paracasei (L. paracasei). Membrane integrity assayed by propidium iodide revealed that approximately 10 % of the L. paracasei cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the L. paracasei culture, fast filtration took several minutes, which is far from ideal for metabolites with high intracellular turnover rates.

Conclusion: The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

No MeSH data available.


Related in: MedlinePlus

The normalized abundance of alanine, glutamic acid, lactic acid and tartaric acid as function of increasing concentrations of glycerol for both alkylation and silylation (n = 5). The data is normalized to the signal intensity obtained with 0 mM of glycerol. A value of 1 indicates that derivatization of the metabolite is unaffected by the increasing concentrations of glycerol
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4559878&req=5

Fig4: The normalized abundance of alanine, glutamic acid, lactic acid and tartaric acid as function of increasing concentrations of glycerol for both alkylation and silylation (n = 5). The data is normalized to the signal intensity obtained with 0 mM of glycerol. A value of 1 indicates that derivatization of the metabolite is unaffected by the increasing concentrations of glycerol

Mentions: Although glycerol saline quenching seems promising for L. paracasei, along with other species [20], it will challenge data acquisition as glycerol cannot be fully eliminated from the final extract (~30 to 50 µL glycerol remains per sample). Non-volatile metabolites require derivatization to make them suitable for GC–MS analysis [34]. The alkylation method employed in this study converts amines and organic acids into volatile esters and carbamates, allowing them to be analysed by GC–MS. Hence, the hydroxyl groups (–OH) in glycerol does not derivatize, and the small amount of glycerol does not affect the GC–MS fingerprints. However, the classical derivatization procedure for metabolome analysis is silylation. Silylation is effective for the analysis of alcohols (including sugars and derivatives), amino acids, organic acids and fatty acids [35]. Evidently, a wider range of metabolites in the metabolome can be analysed. However, the silylation reagent react efficiently with the three hydroxyl groups in glycerol. The effect of increasing concentrations of glycerol on the metabolic fingerprint acquired by alkylation (MCF-GC-MS) and silylation (MSTFA-GC-MS) was evaluated. A highly overloaded glycerol peak was observed in the MSTFA–GC–MS chromatograms causing significant retention time shifts of metabolites (data not shown). The abundance of selected metabolites as function of the increased levels of glycerol is shown in Fig. 4. Fig. 4


Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

Jäpelt KB, Christensen JH, Villas-Bôas SG - Microb. Cell Fact. (2015)

The normalized abundance of alanine, glutamic acid, lactic acid and tartaric acid as function of increasing concentrations of glycerol for both alkylation and silylation (n = 5). The data is normalized to the signal intensity obtained with 0 mM of glycerol. A value of 1 indicates that derivatization of the metabolite is unaffected by the increasing concentrations of glycerol
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4559878&req=5

Fig4: The normalized abundance of alanine, glutamic acid, lactic acid and tartaric acid as function of increasing concentrations of glycerol for both alkylation and silylation (n = 5). The data is normalized to the signal intensity obtained with 0 mM of glycerol. A value of 1 indicates that derivatization of the metabolite is unaffected by the increasing concentrations of glycerol
Mentions: Although glycerol saline quenching seems promising for L. paracasei, along with other species [20], it will challenge data acquisition as glycerol cannot be fully eliminated from the final extract (~30 to 50 µL glycerol remains per sample). Non-volatile metabolites require derivatization to make them suitable for GC–MS analysis [34]. The alkylation method employed in this study converts amines and organic acids into volatile esters and carbamates, allowing them to be analysed by GC–MS. Hence, the hydroxyl groups (–OH) in glycerol does not derivatize, and the small amount of glycerol does not affect the GC–MS fingerprints. However, the classical derivatization procedure for metabolome analysis is silylation. Silylation is effective for the analysis of alcohols (including sugars and derivatives), amino acids, organic acids and fatty acids [35]. Evidently, a wider range of metabolites in the metabolome can be analysed. However, the silylation reagent react efficiently with the three hydroxyl groups in glycerol. The effect of increasing concentrations of glycerol on the metabolic fingerprint acquired by alkylation (MCF-GC-MS) and silylation (MSTFA-GC-MS) was evaluated. A highly overloaded glycerol peak was observed in the MSTFA–GC–MS chromatograms causing significant retention time shifts of metabolites (data not shown). The abundance of selected metabolites as function of the increased levels of glycerol is shown in Fig. 4. Fig. 4

Bottom Line: However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging.The implementation of a reliable, reproducible quenching method is essential within the metabolomics community.Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

View Article: PubMed Central - PubMed

Affiliation: Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. dkkba@chr-hansen.com.

ABSTRACT

Background: Quenching in cold buffered methanol at -40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline.

Results: Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in Lactobacillus paracasei subsp. paracasei (L. paracasei). Membrane integrity assayed by propidium iodide revealed that approximately 10 % of the L. paracasei cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the L. paracasei culture, fast filtration took several minutes, which is far from ideal for metabolites with high intracellular turnover rates.

Conclusion: The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

No MeSH data available.


Related in: MedlinePlus