Comparison of extraction conditions and normalization approaches for cellular metabolomics of adherent growing cells with GC-MS

Hutschenreuther, Antje; Kiontke, Andreas; Birkenmeier, Gerd; Birkemeyer, Claudia

doi:10.1039/c2ay25046b

Cited by 60 publications

(63 citation statements)

References 34 publications

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“…For the adherently growing breast cancer cell line MCF-7 (Michigan Cancer Foundation -7), direct methanolic extraction has showed a suitable extraction efficiency and a high number of detected compounds in the following GC-MS profiling [35]. Application of trypsin to detach adherently growing cells changes the cellular metabolite levels because the enzyme interacts with membrane proteins and consequently alters the physiological state of the cells.…”

Section: Endometabolite Sampling and Gc-ms Profilingmentioning

confidence: 99%

“…Additionally, direct scraping of adherent cells using methanol/water has reported to be the method of choice for harvesting and extraction of cellular metabolites [37,38]. Therefore, we adapted the extraction procedure from Hutschenreuther et al [35] and used ice-cold methanol/water extraction in combination with subsequent cell scraping.…”

Section: Endometabolite Sampling and Gc-ms Profilingmentioning

confidence: 99%

“…Peak intensities have been normalized to the internal standard and blanks have been subtracted. Afterwards normalization to the peak area sum was used as approximation for the averaged number of living cells which cannot be determined due to direct cell scraping[35]. Peak annotation against a mass spectral library resulted in 115 identified compounds (Electronic Supplementary Material…”

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confidence: 99%

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Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines

Willmann

Schlimpert

Halbach

et al. 2015

Journal of Chromatography B

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Section: Endometabolite Sampling and Gc-ms Profilingmentioning

confidence: 99%

Section: Endometabolite Sampling and Gc-ms Profilingmentioning

confidence: 99%

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confidence: 99%

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Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines

Willmann

Schlimpert

Halbach

et al. 2015

Journal of Chromatography B

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“…Breast cancer (BC) is a highly heterogeneous cancer for which morphologically and clinically distinct subgroups of cell lines have been established. 3 Triple negative breast cancer (TNBC) is characterised by the absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor (HER-2). TNBC represents approximately 20% of all BC and is typically associated with poor prognosis.…”

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confidence: 99%

Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231

Kapoore

Coyle

Staton

et al. 2017

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Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (−50°C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism. IntroductionMammalian metabolomics has received increased attention in recent years mainly because of its potential in the prognosis and diagnosis of cancer and for assessing treatment efficacy by analysing cells, fluids or tissues for specific biomarkers in experimental, translational and clinical studies. Adherent mammalian cell-lines are used extensively in oncology research as preclinical models but to date there is insufficient information on the application of metabolomics for the analysis of cultured mammalian cell lines.1,2 To obtain reliable metabolomics data for adherent cells, an optimised workflow must be identified which will provide maximum coverage for all classes of metabolites with minimum leakage. Breast cancer (BC) is a highly heterogeneous cancer for which morphologically and clinically distinct subgroups of cell lines have been established. 3 Triple negative breast cancer (TNBC) is characterised by the absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor (HER-2). TNBC represents approximately 20% of all BC and is typically associated with poor prognosis. Moreover, due to its aggressive phenotype, TNBC only partially res...

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“…These events, generally denoted as matrix effects, result in signal enhancement or suppression and represent thereby an essential challenge in instrumental analyses. 39 Thus, this phenomenon was described for sugars: for example, glucose was reported to enhance the signals of lysine and fumaric and citric acids in plant samples. 19 Here, we observed that sugar-derived signals themselves could be suppressed in the presence of high (100 mmol/L) phosphate concentrations ( Figure 2).…”

Section: ■ Discussionmentioning

confidence: 94%

GC-MS Method for the Quantitation of Carbohydrate Intermediates in Glycation Systems

Milkovska‐Stamenova

Schmidt

Frolov

et al. 2015

J. Agric. Food Chem.

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Glycation is a ubiquitous nonenzymatic reaction of carbonyl compounds with amino groups of peptides and proteins, resulting in the formation of advanced glycation end-products (AGEs) and thereby affecting the properties and quality of thermally processed foods. In this context, mechanisms of the Maillard reaction of proteins need to be understood; that is, glycation products and intermediates (α-dicarbonyls and sugars) need to be characterized. Although the chemical analysis of proteins, peptides, and α-dicarbonyls is well established, sensitive and precise determination of multiple sugars in glycation mixtures is still challenging. This paper presents a gas chromatography-mass spectrometry (GC-MS) method for absolute quantitation of 22 carbohydrates in the model of phosphate-buffered glycation systems. The approach relied on the removal of the phosphate component by polymer-based ion exchange solid phase extraction (SPE) followed by derivatization of carbohydrates and subsequent GC-MS analysis. Thereby, baseline separation for most of the analytes and detection limits of up to 10 fmol were achieved. The method was successfully applied to the analysis of in vitro glycation reactions. Thereby, at least seven sugar-related Maillard reaction intermediates could be identified and quantified. The most abundant reaction product was d-fructose, reaching 2.70 ± 0.12 and 2.38 ± 0.66 mmol/L after 120 min of incubation in the absence and presence of the model peptide, respectively.

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Comparison of extraction conditions and normalization approaches for cellular metabolomics of adherent growing cells with GC-MS

Cited by 60 publications

References 34 publications

Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines

Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines

Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231

GC-MS Method for the Quantitation of Carbohydrate Intermediates in Glycation Systems

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