Extraction parameters for metabolomics from cultured cells

Ser, Zheng; Liu, Xiaojing; Tang, Ngoc Nu; Locasale, Jason W.

doi:10.1016/j.ab.2015.01.003

Cited by 75 publications

(59 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We questioned whether there is any specific molecular mechanism that defines its actions. Using a recently developed high-resolution liquid-chromatography mass spectrometry based technology (Liu et al, 2014; Ser et al, 2015), simultaneous targeted profiling of hundreds of metabolites is possible (Liu et al, 2014). This approach is able to robustly quantify metabolites in concentration ranges over four orders of magnitude, and has been previously applied to study the SGOC network (Mentch et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Targeting One Carbon Metabolism with an Antimetabolite Disrupts Pyrimidine Homeostasis and Induces Nucleotide Overflow

et al. 2016

Self Cite

View full text Add to dashboard Cite

SUMMARY Anti-metabolite agents that affect nucleotide metabolism are frontline chemotherapy agents in several cancers and often successfully target one carbon metabolism. However, the precise mechanisms and resulting determinants of their therapeutic value are unknown. We show that 5-fluorouracil (5-FU), a commonly used anti-metabolite therapeutic with varying efficacy, induces specific alterations to nucleotide metabolism by disrupting pyrimidine homeostasis. An integrative metabolomics analysis of the cellular response to 5-FU shows intracellular uracil accumulation, while deoxyuridine levels exhibited increased flux into the extracellular space resulting in an induction of overflow metabolism. Subsequent analysis from mice bearing colorectal tumors treated with 5-FU, show specific secretion of metabolites in tumor-bearing mice into serum that results from alterations in nucleotide flux and reduction in overflow metabolism. Together these findings identify a determinant of an anti-metabolite response that may be exploited to more precisely define the tumors that could respond to targeting cancer metabolism.

show abstract

Section: Introductionmentioning

confidence: 99%

Targeting One Carbon Metabolism with an Antimetabolite Disrupts Pyrimidine Homeostasis and Induces Nucleotide Overflow

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…29 A further study evaluated the washing protocols with the use of phosphate buffered saline (PBS), water and no washing step for the colorectal cancer cell line HCT116 and reported extraction of metabolites with cold methanol without any washing to be satisfactory. 30 It is important to note that the composition of media used for mammalian cultures are very rich in nutrients such as amino acids, vitamins, sugars, which are also present inside the cells as intracellular metabolites. Hence subsequent extraction of cells followed by quenching, without any washing step might result in false higher recoveries for specific intracellular metabolites that are found to be present in the medium as well.…”

mentioning

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

Analyst

View full text Add to dashboard Cite

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...

show abstract

“…Rat brains were homogenized using a potter in 1 ml of pre-chilled methanol/water 1:1 solution, containing 10 nmol of internal standard, and centrifuged at 10,000 g for 10 min at 4 • C (Ser et al, 2015). The resulting supernatants were collected and transferred into Eppendorf tubes and stored at −80 • C. Analyses were performed according to a previous protocol (Sommella et al, 2019).…”

Section: Mass Spectrometry-based Metabolomics Statistics and Analysismentioning

confidence: 99%

The Pomace Extract Taurisolo Protects Rat Brain From Ischemia-Reperfusion Injury

Lapi

Stornaiuolo

Sabatino

et al. 2020

Front. Cell. Neurosci.

View full text Add to dashboard Cite

Taurisolo is a pomace extract from Aglianico Grapes, a wine cultivar native to Campania (Southern Italy). It exhibits a very high polyphenolic content and, consumed as a nutraceutical, is effective in reducing the level of Trimethylamine N-oxide (TMAO), a cardiovascular disease risk factor marker. We here show the effects of Taurisolo on rat brain microvascular alterations induced by a diminution in cerebral blood flow (CBFD) for 30 min, due to bilateral common carotid artery occlusion, and subsequent blood flow restoration (CBFR) for 60 min. The rat pial microcirculation was investigated by intravital fluorescence microscopy through a parietal closed window implanted into the skull bone. The rat pial arterioles were classified according to Strahler's ordering scheme, from smaller penetrating arterioles up to the larger ones. Western blotting analysis and mass spectrometry (MS)-based metabolomics were used to investigate the expression of endothelial nitric oxide synthase (eNOS) or the presence of peroxidized cardiolipin and several inflammatory mediators, respectively. Radical Oxygen Species (ROS) formation and neuronal loss were assessed. In rats CBFD and CBFR caused a decrease in arteriolar diameter, increase in fluorescent leakage and in adhesion of leukocytes to venular walls, reduction in the length of perfused capillaries and increment of ROS formation with large infarct size. Taurisolo , intravenously or orally administered, induced pial arteriolar dilation (up to >30% of baseline), prevented fluorescent leakage, adhesion of leukocytes, ROS formation, while facilitated capillary perfusion and significantly reduced infarct size. These effects were accompanied by an increase in eNOS expression. Mass-spectrometry metabolomics analysis detected a marked decrease in the amount

show abstract

Extraction parameters for metabolomics from cultured cells

Cited by 75 publications

References 28 publications

Targeting One Carbon Metabolism with an Antimetabolite Disrupts Pyrimidine Homeostasis and Induces Nucleotide Overflow

Targeting One Carbon Metabolism with an Antimetabolite Disrupts Pyrimidine Homeostasis and Induces Nucleotide Overflow

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

The Pomace Extract Taurisolo Protects Rat Brain From Ischemia-Reperfusion Injury

Contact Info

Product

Resources

About