Reducing Time and Increasing Sensitivity in Sample Preparation for Adherent Mammalian Cell Metabolomics

Departments of Chemistry, The University of Michigan, Ann Arbor, 48109, United States.
Analytical Chemistry (Impact Factor: 5.64). 04/2011; 83(9):3406-14. DOI: 10.1021/ac103313x
Source: PubMed


A simple, fast, and reproducible sample preparation procedure was developed for relative quantification of metabolites in adherent mammalian cells using the clonal β-cell line INS-1 as a model sample. The method was developed by evaluating the effect of different sample preparation procedures on high performance liquid chromatography- mass spectrometry quantification of 27 metabolites involved in glycolysis and the tricarboxylic acid cycle on a directed basis as well as for all detectable chromatographic features on an undirected basis. We demonstrate that a rapid water rinse step prior to quenching of metabolism reduces components that suppress electrospray ionization thereby increasing signal for 26 of 27 targeted metabolites and increasing total number of detected features from 237 to 452 with no detectable change of metabolite content. A novel quenching technique is employed which involves addition of liquid nitrogen directly to the culture dish and allows for samples to be stored at -80 °C for at least 7 d before extraction. Separation of quenching and extraction steps provides the benefit of increased experimental convenience and sample stability while maintaining metabolite content similar to techniques that employ simultaneous quenching and extraction with cold organic solvent. The extraction solvent 9:1 methanol: chloroform was found to provide superior performance over acetonitrile, ethanol, and methanol with respect to metabolite recovery and extract stability. Maximal recovery was achieved using a single rapid (∼1 min) extraction step. The utility of this rapid preparation method (∼5 min) was demonstrated through precise metabolite measurements (11% average relative standard deviation without internal standards) associated with step changes in glucose concentration that evoke insulin secretion in the clonal β-cell line INS-1.

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    • "The use of acetone for de-proteinization of mammalian cells prior to metabolome profiling has also been suggested for both suspension cell cultures (Aranibar et al. 2011; Bruce et al. 2009; Tiziani et al. 2008) and adherently grown cells (Danielsson et al. 2010). Recently, the use of liquid nitrogen for direct quenching, with subsequent extraction using a suitable solvent, was suggested for LC–MS based metabolomics of adherently grown clonal b-cell line INS-1 (Lorenz et al. 2011) and human pancreatic cancer cells Panc-1 (Bi et al. 2013). Interestingly , the breast cancer MCF-7 cell line has been analysed using GC–MS based metabolomics after a similar quenching and extraction approach (Hutschenreuther et al. 2012). "
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    ABSTRACT: Analysis of the metabolome can be sample and cell dependent. In this study, we compared two conventional pre-treatment approaches (trypsinization and cell scraping) in three adherently grown mammalian cell lines (two breast cancer cell lines MDA-MB-436, MCF-7 and an endothelial cell line—HMEC-1). We report experimental evidence, for the first time, demonstrating that metabolite leakage occurs with both treatments, and that the cell lines are differentially influenced. In addition, we examined two recently reported approaches of simultaneous quenching and extraction that showed minimal metabolome leakage. We also investigated the culture of cells on beads for rapid quenching and extraction, as a novel sample handling protocol. For metastatic breast cancer cells MDA-MB-436, the two direct quenching approaches and the bead harvesting approach showed favourable results with respect to metabolome leakage, compared to the conventional approaches. We characterised the recovery of eleven different classes of metabolites identified by gas chromatography–mass spectrometry in the cell extracts and the supernatants following quenching. Analysis of results based on metabolite classes is shown to be a useful approach aiding metabolomic interpretations. We also examined the effect of including a protein precipitation step on the metabolite classes detected. The de-proteinization step did not show significant improvement in overall recoveries. This investigation suggests that it is important to establish the level of metabolome leakage for the specific cell line investigated, irrespective of the methodology employed. Rapid approaches that combine quenching and extraction steps may be more effective in retaining valid metabolome data, with minimal metabolome leakage occurring.
    Metabolomics 07/2015; DOI:10.1007/s11306-015-0833-4 · 3.86 Impact Factor
    • "Some recent works have claimed the benefits of combining in a single step quenching and extraction procedures in mammalian adherent cells [23] [24] "
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    ABSTRACT: In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5'-deoxy-5'-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed. Copyright © 2015 Elsevier B.V. All rights reserved.
    Journal of Pharmaceutical and Biomedical Analysis 03/2015; 110. DOI:10.1016/j.jpba.2015.03.001 · 2.98 Impact Factor
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    • "Despite the existence of standardised sample preparation methodologies (Folch et al., 1957; Hara & Radin, 1978; Wu et al., 2008), problems may arise with specific organisms , in the case of insect studies this is often specifically related to low biomass (Lorenz et al., 2011; Marcinowska et al., 2011; Kim et al., 2013). As the majority of extraction methodologies are tailored for larger biomass samples, the volumes and ratios associated with these approaches may "
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    ABSTRACT: Metabolomic analyses can reveal associations between an organism's metabolome and further aspects of its phenotypic state, an attractive prospect for many life-sciences researchers. The metabolomic approach has been employed in some, but not many, insect study systems, starting in 1990 with the evaluation of the metabolic effects of parasitism on moth larvae. Metabolomics has now been applied to a variety of aspects of insect biology, including behaviour, infection, temperature stress responses, CO2 sedation, and bacteria–insect symbiosis. From a technical and reporting standpoint, these studies have adopted a range of approaches utilising established experimental methodologies. Here, we review current literature and evaluate the metabolomic approaches typically utilised by entomologists. We suggest that improvements can be made in several areas, including sampling procedures, the reduction in sampling and equipment variation, the use of sample extracts, statistical analyses, confirmation, and metabolite identification. Overall, it is clear that metabolomics can identify correlations between phenotypic states and underlying cellular metabolism that previous, more targeted, approaches are incapable of measuring. The unique combination of untargeted global analyses with high-resolution quantitative analyses results in a tool with great potential for future entomological investigations.
    Entomologia Experimentalis et Applicata 02/2015; 155(1). DOI:10.1111/eea.12281 · 1.62 Impact Factor
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