Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

Teleki, Attila; Santiago, Andrés; Takors, Ralf

doi:10.1016/j.ab.2015.01.002

Cited by 73 publications

(68 citation statements)

References 60 publications

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“…The resulting data sets were analyzed with MassHunter B.04.00 software. Analysis was based on a previously described isotope dilution mass spectrometry (IDMS) method using 13 C‐labeled internal standard which was extracted from U‐ 13 C labeled algal cells (>99 atom% 13 C, lot no. 487945, Sigma‐Aldrich, Taufkirchen, Germany) .…”

Section: Methodsmentioning

confidence: 99%

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Pfizenmaier

Junghans

Teleki

2016

Biotechnology Journal

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Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed-batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg(∑1) ) resulting from feeding of nutrients may enhance specific product formation rates (qp ). As an improved ATP supply was anticipated to enhance qp this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg(-1) ). Hyperosmotic culture conditions caused a ∼2.6-fold increase of specific ATP formation rates together with a ∼1.7-fold rise in cytosolic and mitochondrial ATP-pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR-132-3p, miR-132-5p, miR-182, miR-183, miR-194, miR-215-3p, miR-215-5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR-183 may be the most promising target to enhance qp by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.

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Section: Methodsmentioning

confidence: 99%

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Pfizenmaier

Junghans

Teleki

2016

Biotechnology Journal

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“…With the exception of pyruvate all other intracellular metabolites were absolutely quantified with the help of a newly developed liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) method [31] and internal standards in 13 C labeled algae [32]. Analytes were measured by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a triple quadrupol instrument (Agilent Technologies 6410B).…”

Section: Analytics Of Cell Extractsmentioning

confidence: 99%

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Matuszczyk

Teleki

Pfizenmaier

2015

Biotechnology Journal

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Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells. The approach consists of a subtracting filtering method separating cytosolic components from physically intact mitochondrial compartments. The internal standards glucose-6-phosphate and cis-aconitate were chosen to quantify cytosolic secession and mitochondrial membrane integrity. Extracts of related fractions were studied by liquid chromatography-isotope dilution mass spectrometry for the absolute quantification of a subset of glycolytic and tricarboxylic acid cycle intermediates together with the adenylate nucleotides ATP, ADP and AMP. The application of the protocol revealed highly dynamic changes in the related pool sizes as a function of distinct cultivation periods of IgG1 producing CHO cells. Mitochondrial and cytosolic pool dynamics were in agreement with anticipated metabolite pools of independent studies. The analysis of adenosine phosphate levels unraveled significantly higher ATP levels in the cytosol leading to the hypothesis that mitochondria predominantly serve for fueling ATP into the cytosol where it is tightly controlled at physiological adenylate energy charges about 0.9.

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“…More recently developed ion‐pair reversed‐phase LC–MS methods yielded LODs in the range from 1 to 10 nM for AMP, ADP, and ATP using multiple reaction monitoring and an injection volume of 5 µL . HILIC–MS‐based approaches recently developed for nucleotide profiling in various biological samples provided LODs typically in the range from 2 to 100 nM using MS/MS mode and employing an injection volume of 5 µL . In this context, the proposed sheathless CZE–MS method (used in full scan MS mode only) provided concentration LODs for the studied nucleotides that are comparable to or better than LODs obtained by LC–MS‐based approaches employing multiple reaction monitoring.…”

Section: Resultsmentioning

confidence: 99%

Profiling nucleotides in low numbers of mammalian cells by sheathless CE–MS in positive ion mode: Circumventing corona discharge

et al. 2020

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Negative ion mode nano‐ESI–MS is often considered for the analysis of acidic compounds, including nucleotides. However, under high aqueous separation conditions, corona discharge is frequently observed at emitter tips, which may result in low ion abundances and reduced nano‐ESI needle emitter lifetimes. In this work, we introduce a sheathless CE‐MS method for the highly efficient and sensitive analysis of nucleotides employing ESI in positive ion mode, thereby fully circumventing corona discharge. By using a background electrolyte of 16 mM ammonium acetate (pH 9.7) a mixture of 12 nucleotides, composed of mono‐, di‐, and tri‐phosphates, could be efficiently analyzed with plate numbers per meter above 220 000 and with LODs in the range from 0.06 to 1.3 nM, corresponding to 0.4 to 8.6 attomole, when using an injection volume of about 6.5 nL only. The utility of the method was demonstrated for the profiling of nucleotides in low numbers of mammalian cells using HepG2 cells as a model system. Endogenous nucleotides could be efficiently analyzed in extracts from 50 000 down to 500 HepG2 cells only. Moreover, apart from nucleotides, also some nicotinamide‐adenine dinucleotides and amino acids could be analyzed under these conditions, thereby clearly illustrating the utility of this approach for metabolic profiling of low amounts of biological material.

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Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

Cited by 73 publications

References 60 publications

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Profiling nucleotides in low numbers of mammalian cells by sheathless CE–MS in positive ion mode: Circumventing corona discharge

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