Determination of the phosphorylated sugars of the Embden-Meyerhoff-Parnas pathway inLactococcus lactis using a fast sampling technique and solid phase extraction

Jensen, Niels Bang Siemsen; Jokumsen, Kirsten Væver; Villadsen, John

doi:10.1002/(sici)1097-0290(19990505)63:3<356::aid-bit12>3.0.co;2-1

Cited by 48 publications

(8 citation statements)

References 11 publications

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“…Quenching in cold methanol has since become the preferred method for quenching microorganisms in metabolic fingerprinting studies [ 29 , 30 ]. However, in contrast to yeast cells, intracellular metabolites have been shown to leak from bacterial cells when brought into contact with cold methanol [ 5 , 8 , 19 , 23 , 31 ]. Alternative quenching strategies that attempt to stabilise the bacterial cell during methanol quenching have been tested with mixed success.…”

Section: Introductionmentioning

confidence: 99%

Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy

2015

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BackgroundQuenching 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.ResultsOur 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.ConclusionThe 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.

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Section: Introductionmentioning

confidence: 99%

Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy

2015

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“…They concluded that S. cerevisiae cells do not leak metabolites when quenched in 60% methanol at −40°C [2], and this has also been supported by several other studies [3], [4]. The situation is different for bacteria, as there have been numerous reports of metabolite leakage of intracellular metabolites from bacterial cells during the quenching process [5], [6], [7]. Bolten et al .…”

Section: Introductionmentioning

confidence: 76%

The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

et al. 2011

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Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris.

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“…Bolten and co-workers (2007), for example, showed a 90% reduction in the concentration of free amino acids when quenching microbial cells with cold-methanol solution [ 13 ]. Variants of the original method proposed by de Koning and van Dam (1992) have been proposed for quenching yeast and bacterial cells [ 13 , 38 , 40 , 52 , 53 ]. Although some satisfactory results were observed, the use of cold-methanol solution for quenching the metabolism of microbial cells remains controversial.…”

Section: Overview Of Available Quenching Methods For Microbial Culmentioning

confidence: 99%

“…Analysts must be aware that bacterial cells are particularly sensitive to cold shock and that intracellular metabolites may leak when the cells are subjected to quick changes in temperature [ 41 , 53 ]. According to Leder (1972), the cold-shock phenomenon can be prevented or minimized by a simultaneous hyperosmotic transition [ 59 ].…”

Section: Overview Of Available Quenching Methods For Microbial Culmentioning

confidence: 99%

Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

2017

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Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

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Determination of the phosphorylated sugars of the Embden-Meyerhoff-Parnas pathway inLactococcus lactis using a fast sampling technique and solid phase extraction

Cited by 48 publications

References 11 publications

Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy

Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy

The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

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