Optimization of cellular nucleotide extraction and sample preparation for nucleotide pool analyses using capillary electrophoresis

Grob, Miriam; O'Brien, Kylie B.; Chu, Juan Jua; Chen, David D. Y.

doi:10.1016/s1570-0232(02)01033-4

Cited by 33 publications

(31 citation statements)

References 25 publications

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“…Previously, Grob et al, (2003), also concluded 20 to 60% acetonitrile in water is the best solvent for extracting nucleotides in mouse lymphoma cells.…”

Section: Metabolite Extractionmentioning

confidence: 98%

“…Furthermore, extraction solvents should be compatible with the subsequent analytical procedures (Villas-Boas et al, 2005b). A wide range of different extraction solutions have been reported including alcohols, water, acids, bases and organic solvents at high or low temperature (de Koning and van Dam, 1992;Dietmair et al, 2010;Grob et al, 2003;Maharjan and Ferenci, 2003;Ritter et al, 2008;Sellick et al, 2009;Shryock et al, 1986;Villas-Boas et al, 2005a;Villas-Boas et al, 2005b;, and each solution is only suitable for specific classes of metabolites and certain cell types (Canelas et al, 2009;de Koning and van Dam, 1992;Grob et al, 2003;Maharjan and Ferenci, 2003;Rabinowitz and Kimball, 2007;Shryock et al, 1986;Tomiya et al, 2001;Villas-Boas et al, 2005b). Therefore, the extraction protocol is selected depending on the aim of study and the target biological sample (Alvarez-Sanchez, 2010), as well as being based on the nature and level of metabolites extracted (Dunn and Ellis, 2005).…”

Section: Metabolite Extractionmentioning

confidence: 99%

“…Protocols for intracellular metabolite extraction and measurement have been reported widely for bacteria Winder et al, 2008;Wittmann et al, 2004;Wu et al, 2010), yeast Loret et al, 2007;Villas-Boas et al, 2005a) and mammalian cells Grob et al, 2003;Ritter et al, 2008;Sellick et al, 2010). To our knowledge, however, no such protocol has been validated for insect cell cultures, especially baculovirus infected insect cells.…”

Section: Intracellular Metabolites Extraction Procedures For Insect Cellsmentioning

confidence: 99%

“…Sample preparation protocols for endometabolome analysis have been developed for yeast [14][15][16], bacteria [17][18][19][20] and mammalian cells [11][12][13]21,22], but as yet not for insect cells. Dietmair et al developed an optimized methodology for quantitative metabolomics of Chinese Hamster Ovary (CHO) cells, using ice-cold isotonic NaCl (0.9% w/v) and very cold 50% v/v acetonitrile (À40°C) for effective quenching and extraction of intracellular metabolites, respectively [12].…”

Section: Introductionmentioning

confidence: 99%

“…Whereas, there are comparatively few papers comparing quenching and extraction methods in mammalian cells Grob et al, 2003;Sellick et al, 2009;Sellick et al, 2010). Ritter et al, (2008) Previously, Grob et al, (2003), also concluded 20 to 60% acetonitrile in water is the best solvent for extracting nucleotides in mouse lymphoma cells. …”

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confidence: 99%

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Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

Tran¹

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The baculovirus insect cell system has been used widely for biopesticide applications and for the production of recombinant proteins, vaccines and vectors for gene delivery. To be commercially viable, especially for the low margin applications of biopesticides and veterinary vaccines, the yield of this production system needs to be improved. One of the important approaches is the infection of insect cell cultures at high cell densities in order to maximize volumetric production. However, the cell specific yield gradually reduces with increasing infection cell density (ICD). As a result, the volumetric production can not improve further regardless of efforts to increase the uninfected cell density in batch or fedbatch cultures. The phenomenon known as "the cell density effect" appears not only during the late stage of protein production, but also during the early stage of virus replication and transcription. Discovering the changes in intra and extracellular metabolites between low and high ICDs may reveal evidence for improving yields. This PhD thesis focuses on the development of an intracellular metabolite extraction protocol for infected insect cell cultures and for measuring intra and extracellular metabolites of infected cells of different baculovirus infected insect cell systems.Prior to analyzing the intracellular metabolites of low and high ICDs, an extraction protocol was developed and validated for insect cell cultures, especially infected insect cells. The ice-cold saline quenching solution developed for mammalian cells did not work well for insect cells, especially for infected cultures. Therefore, a modification of the quenching solution was made by an addition of 0.2% Pluronic® F-68 that successfully protected the cell membrane during quenching and washing procedures. In addition, one wash was found to be enough for removal of medium originated metabolites adhering on the surface of cell membranes. The ATP recovery (over the direct extract) of the extraction protocol using ice-cold saline quenching solution plus 0.2% Pluronic® F-68 after one wash was almost 90% for Spodoptera frugiperda (Sf9) cells infected with a recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV) and Helicoverpa zea (HzAM1) cells infected with a wild-type Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) at 24 hours post infection (hpi).The intracellular metabolite patterns and substrate consumption rates of low versus high ICDs was first investigated for the Sf9/rAcMNPV system in Sf900™III medium. A preliminary study was conducted to identify how late post-infection the cell membrane integrity was maintained during the quenching and washing processes. The result showed that the extraction protocol is efficient up to 48 hpi for infected Sf9 cells. Levels of intracellular nucleotides (tri-phosphates), amino acids (AA), and TCA cycle intermediates iii (α-ketoglutarate, pyruvate) during an infection at a low ICD (2×10 6 cells/mL) were shown to be higher than those during an infection at a high...

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“…Previously, Grob et al, (2003), also concluded 20 to 60% acetonitrile in water is the best solvent for extracting nucleotides in mouse lymphoma cells.…”

Section: Metabolite Extractionmentioning

confidence: 98%

Section: Metabolite Extractionmentioning

confidence: 99%

Section: Intracellular Metabolites Extraction Procedures For Insect Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

Tran¹

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Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse

Cachaço

Pereira

Pardal

et al. 2005

Developmental Dynamics

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Here, we determined ␣ subunit expression patterns at embryonic day (E) 11.5-E14.5. Differentiated myotomal myocytes express all ␣ subunits studied. As the muscle masses form both in trunk (E12.5) and limbs (E11.5-E12.5), laminin receptors ␣6␤1 and ␣7␤1 are undetectable, and an assembled laminin matrix is absent. Instead ␣1␤1, ␣4␤1, ␣5␤1, and an ␣v-containing integrin are expressed and unassembled laminin and fibronectin are abundant around myogenic cells. At E13.5-E14.5, ␣6␤1 and ␣7␤1 are expressed, and a laminin matrix forms around individual myotubes. Thus, myogenic cells change their integrin expression pattern during the course of primary myogenesis in the mouse, suggesting different roles for fibronectin-and laminin-containing matrices in this process.

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In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography

Kulp¹,

Kaljurand²,

Käämbre

et al. 2004

Electrophoresis

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A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.

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Optimization of cellular nucleotide extraction and sample preparation for nucleotide pool analyses using capillary electrophoresis

Cited by 33 publications

References 25 publications

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography

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