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: 5.83). 04/2011; 83(9):3406-14. DOI: 10.1021/ac103313x
Source: PubMed

ABSTRACT 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.

  • Source
    • "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 "
    [Show abstract] [Hide abstract]
    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 · 1.71 Impact Factor
  • Source
    • "The extraction protocols, which could be optimised to give higher yields and better repeatability for metabolite isolation, are clearly cell-type dependent, and many procedures have been described for plants, bacteria and yeasts (Faijes et al., 2007; Gromova and Roby, 2010; Sekiyama et al., 2011). The lysis of adherent mammalian cells is more complicated because of the difficulty in isolating the sample (Lorenz et al., 2011; Martineau et al., 2011). According to previous protocols, washing and quenching steps are suggested to eliminate residual cellculture media and to stop metabolite evolution before extraction . "
    [Show abstract] [Hide abstract]
    ABSTRACT: As a complement to the classic metabolomics biofluid studies, the visualisation of the metabolites contained in cells or tissues could be a very powerful tool to understand how the local metabolism and biochemical pathways could be affected by external or internal stimuli or pathologies. Therefore, extraction and/or lysis is necessary to obtain samples adapted for use with the current analytical tools (liquid NMR and MS). These extraction or lysis work-ups are often the most labour-intensive and rate-limiting steps in metabolomics, as they require accuracy and repeatability as well as robustness. Many of the procedures described in the literature appear to be very time-consuming and not easily amenable to automation. To find a fast, simplified procedure that allows release of the metabolites from cells and tissues in a way that is compatible with NMR analysis. We assessed the use of sonication to disrupt cell membranes or tissue structures. Both a vibrating probe and an automated bath sonicator were explored. The application of sonication as the disruption procedure led to reproducible NMR spectral data compatible with metabolomics studies. This method requires only a small biological tissue or cell sample, and a rapid, reduced work-up was applied before analysis. The spectral patterns obtained are comparable with previous, well-described extraction protocols. The rapidity and the simplicity of this approach could represent a suitable alternative to the other protocols. Additionally, this approach could be favourable for high- throughput applications in intracellular and intratissular metabolite measurements. Copyright © 2014 John Wiley & Sons, Ltd.
    Phytochemical Analysis 07/2014; 25(4). DOI:10.1002/pca.2498 · 2.45 Impact Factor
  • Source
    • "However, there is no standardised method for metabolite sample preparation and the isolation of metabolites requires different SPE protocols guided by the compound class the researcher is interested in [52]. Alternative sample preparation methods very often are differing from the standard RP separation used in a proteomics laboratory [53] [54]. Next to GC-MS, CE-MS and NMR, LC-MS has emerged as a major analytical platform for metabolite analysis. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proteomic research facilities and laboratories are facing increasing demands for the integration of biological data from multiple "-OMICS" approaches. The aim to fully understand biological processes requires the integrated study of genomes, proteomes and metabolomes. While genomics and proteomic workflows are different, the study of the metabolome overlaps significantly with the latter, both in instrumentation and methodology. However, chemical diversity complicates an easy and direct access to the metabolome by mass spectrometry. The present review provides an introduction into metabolomics workflows from the viewpoint of proteomic researchers. We compare the physicochemical properties of proteins and peptides with metabolites/small molecules to establish principle differences between these analyte classes based on human data. We highlight the implications this may have on sample preparation, separation, ionisation, detection and data analysis. We argue that a typical proteomic workflow (nLC-MS) can be exploited for the detection of a number of aliphatic and aromatic metabolites, including fatty acids, lipids, prostaglandins, di-/tri-peptides, steroids and vitamins, thereby providing a straight-forward entry point for metabolomics-based studies. Limitations and requirements are discussed as well as extensions to the standard workflow to expand the range of detectable molecular classes without investing into dedicated instrumentation such as GC-MS, CE-MS or NMR. This article is protected by copyright. All rights reserved.
    Proteomics 12/2013; 13(23-24). DOI:10.1002/pmic.201300192 · 3.97 Impact Factor
Show more


1 Download
Available from