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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 metabolic fingerprints (TICs) of L. paracasei for the cold buffered methanol and glycerol saline quenching, in the retention time region from 5.6 to 35.0 min. All TICs are acquired using MCF–GC–MS, and the metabolites identified using an in-house MS library. The TIC for cold buffered methanol is inverted to ease interpretation
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Fig2: The metabolic fingerprints (TICs) of L. paracasei for the cold buffered methanol and glycerol saline quenching, in the retention time region from 5.6 to 35.0 min. All TICs are acquired using MCF–GC–MS, and the metabolites identified using an in-house MS library. The TIC for cold buffered methanol is inverted to ease interpretation

Mentions: The loss of cell membrane integrity during cold buffered methanol quenching is expected to introduce a negative bias (i.e. underestimation of intracellular metabolites), and a lower precision of the metabolic fingerprint (i.e. increased variability). The MCF–GC–MS total ion chromatograms (TICs) after quenching with cold buffered methanol and cold glycerol saline are shown in Fig. 2. A total of 58 ± 4 and 64 ± 2 metabolites were identified for cold buffered methanol and glycerol saline (n = 6), respectively. The identities of some of the major peaks are marked in Fig. 2. A number of the peaks, marked with asterisks, originated from glycerol attached to the cells. Glycerol contains organic impurities, and these should be excluded from further analysis [20].Fig. 2


Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

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

The metabolic fingerprints (TICs) of L. paracasei for the cold buffered methanol and glycerol saline quenching, in the retention time region from 5.6 to 35.0 min. All TICs are acquired using MCF–GC–MS, and the metabolites identified using an in-house MS library. The TIC for cold buffered methanol is inverted to ease interpretation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: The metabolic fingerprints (TICs) of L. paracasei for the cold buffered methanol and glycerol saline quenching, in the retention time region from 5.6 to 35.0 min. All TICs are acquired using MCF–GC–MS, and the metabolites identified using an in-house MS library. The TIC for cold buffered methanol is inverted to ease interpretation
Mentions: The loss of cell membrane integrity during cold buffered methanol quenching is expected to introduce a negative bias (i.e. underestimation of intracellular metabolites), and a lower precision of the metabolic fingerprint (i.e. increased variability). The MCF–GC–MS total ion chromatograms (TICs) after quenching with cold buffered methanol and cold glycerol saline are shown in Fig. 2. A total of 58 ± 4 and 64 ± 2 metabolites were identified for cold buffered methanol and glycerol saline (n = 6), respectively. The identities of some of the major peaks are marked in Fig. 2. A number of the peaks, marked with asterisks, originated from glycerol attached to the cells. Glycerol contains organic impurities, and these should be excluded from further analysis [20].Fig. 2

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