Cleanup and Analysis of Sugar Phosphates in Biological Extracts by Using Solid-Phase Extraction and Anion-Exchange Chromatography with Pulsed Amperometric Detection

Smits, Hans Peter; Cohen, Arieh; Buttler, Torbjörn; Nielsen, Jens; Olsson, Lisbeth

doi:10.1006/abio.1998.2714

Cited by 73 publications

(67 citation statements)

References 32 publications

(26 reference statements)

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“…Measurement of Intracellular Metabolites-The cell extracts from aerobic batch cultivations and cleanup of sugar phosphates (Gal-1-P, Glu-1-P, Glu-6-P, Man-6-P, and Fru-6-P), were obtained by applying a method described previously (24). Cell extracts were obtained using cold methanol as quenching agent and chloroform as extraction solvent.…”

Section: Methodsmentioning

confidence: 99%

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Transcriptional, Proteomic, and Metabolic Responses to Lithium in Galactose-grown Yeast Cells

Bro

Regenberg

Lagniel³

et al. 2003

Journal of Biological Chemistry

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Lithium is highly toxic to yeast when grown in galactose medium mainly because phosphoglucomutase, a key enzyme of galactose metabolism, is inhibited. We studied the global protein and gene expression profiles of Saccharomyces cerevisiae grown in galactose in different time intervals after addition of lithium. These results were related to physiological studies where both secreted and intracellular metabolites were determined. Microarray analysis showed that 664 open reading frames were down-regulated and 725 up-regulated in response to addition of lithium. Genes involved in transcription, translation, and nucleotide metabolism were down-regulated at the transcriptional level, whereas genes responsive to different stresses as well as genes from energy reserve metabolism and monosaccharide metabolism were up-regulated. Compared with the proteomic data, 26% of the down-regulated and 48% of the up-regulated proteins were also identified as being changed on the mRNA level. Functional clusters obtained from proteome data were coincident with transcriptional clusters. Physiological studies showed that acetate, glycerol, and glycogen accumulate in response to lithium, as reflected in expression data, whereas a change from respiro-fermentative to respiratory growth could not be predicted from the expression analyses.

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Section: Methodsmentioning

confidence: 99%

“…Cell extracts were obtained using cold methanol as quenching agent and chloroform as extraction solvent. Furthermore, cleanup of sugar phosphates was done using solid-phase extraction (24). Determination of sugar phosphates was done by anionexchange chromatography with pulsed amperometric detection as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional, Proteomic, and Metabolic Responses to Lithium in Galactose-grown Yeast Cells

Bro

Regenberg

Lagniel³

et al. 2003

Journal of Biological Chemistry

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“…Sugars, sugar phosphates, sugar alcohols and peptides were determined simultaneously by GC-MS after methoxymation and silylation derivatization, following the protocol described by Roessner et al (2000). The determination of extracellular sugar phosphates for the evaluation of leakage during quenching was performed using solid-phase extraction and high-pressure ion-exchange chromatography with pulsed amperometric detection (HPIC-PAD), according to Smits et al (1998).…”

Section: Analytical Proceduresmentioning

confidence: 99%

“…Two different inlet inserts were used, depending on the derivatives to be analysed: Siltek gooseneck splitless deactivated liner (Restek, USA), 4.0 mm i.d., was used for analysis of MCF derivatives, and split straight liner with glasswool (Agilent Technologies, USA) for the TMS-derivatives. HPIC-PAD analysis was performed with a Dionex BioLC (Dionex, Sunnyvale, CA, USA) and the analytes were separated in a CarboPac PA1 column (Dionex) and a Dionex PA1 guard column at 30 • C, under conditions described by Smits et al (1998).…”

Section: Analytical Proceduresmentioning

confidence: 99%

“…Their method has recently been applied for intracellular metabolite analysis of microorganisms (Cremin et al, 1995;Ruijter and Visser, 1996;Smits et al, 1998;Jensen et al, 1999;Zaldivar et al, 2002;Maharjan and Ferenci, Sample preparation for global metabolite analysis 1157 2003) and animal tissues (Le Belle et al, 2002). The method presents good recovery of phosphorylated compounds (Smits et al, 1998;Jensen et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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Global metabolite analysis of yeast: evaluation of sample preparation methods

Villas‐Bôas

Højer-Pedersen

Åkesson

et al. 2005

Yeast

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364

363

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Sample preparation is considered one of the limiting steps in microbial metabolome analysis. Eukaryotes and prokaryotes behave very differently during the several steps of classical sample preparation methods for analysis of metabolites. Even within the eukaryote kingdom there is a vast diversity of cell structures that make it imprudent to blindly adopt protocols that were designed for a specific group of microorganisms. We have therefore reviewed and evaluated the whole sample preparation procedures for analysis of yeast metabolites. Our focus has been on the current needs in metabolome analysis, which is the analysis of a large number of metabolites with very diverse chemical and physical properties. This work reports the leakage of intracellular metabolites observed during quenching yeast cells with cold methanol solution, the efficacy of six different methods for the extraction of intracellular metabolites, and the losses noticed during sample concentration by lyophilization and solvent evaporation. A more reliable procedure is suggested for quenching yeast cells with cold methanol solution, followed by extraction of intracellular metabolites by pure methanol. The method can be combined with reduced pressure solvent evaporation and therefore represents an attractive sample preparation procedure for high-throughput metabolome analysis of yeasts.

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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

Jensen

Jokumsen

Villadsen

1999

Biotechnol. Bioeng.

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An experimental procedure for the determination of intracellular concentrations of the phosphorylated sugars in the lactic acid bacterium Lactococcus lactis is presented. The first step of the procedure is a rapid sampling of a small volume of the growth medium into 60% (v/v) methanol precooled to −35°C, bringing about a fast and complete stop of all metabolic activity. In contrast to yeast the metabolites leak out of the cells when these are brought into contact with methanol and are present in the medium and in the biomass after the quenching. A liquid–liquid extraction with chloroform at −25°C ensures a total permeability of the cellular membrane towards the metabolites of interest as well as the inactivation of enzymes liable to alter their levels. The final step of the procedure consists in a solid phase extraction using columns with a high affinity for phosphorylated components. The internal standard was recovered to an extent of 85–95%. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 356–362, 1999.

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Cleanup and Analysis of Sugar Phosphates in Biological Extracts by Using Solid-Phase Extraction and Anion-Exchange Chromatography with Pulsed Amperometric Detection

Cited by 73 publications

References 32 publications

Transcriptional, Proteomic, and Metabolic Responses to Lithium in Galactose-grown Yeast Cells

Transcriptional, Proteomic, and Metabolic Responses to Lithium in Galactose-grown Yeast Cells

Global metabolite analysis of yeast: evaluation of sample preparation methods

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

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