Procedure for tissue sample preparation and metabolite extraction for high-throughput targeted metabolomics
- 5.5k Downloads
Reproducible quantification of metabolites in tissue samples is of high importance for characterization of animal models and identification of metabolic changes that occur in different tissue types in specific diseases. However, the extraction of metabolites from tissue is often the most labor-intensive and error-prone step in metabolomics studies. Here, we report the development of a standardized high-throughput method for rapid and reproducible extraction of metabolites from multiple tissue samples from different organs of several species. The method involves a bead-based homogenizer in combination with a simple extraction protocol and is compatible with state-of-the-art metabolomics kit technology for quantitative and targeted flow injection tandem mass spectrometry. We analyzed different extraction solvents for both reproducibility as well as suppression effects for a range of different animal tissue types including liver, kidney, muscle, brain, and fat tissue from mouse and bovine. In this study, we show that for most metabolites a simple methanolic extraction is best suited for reliable results. An additional extraction step with phosphate buffer can be used to improve the extraction yields for a few more polar metabolites. We provide a verified tissue extraction setup to be used with different indications. Our results demonstrate that this high-throughput procedure provides a basis for metabolomic assays with a wide spectrum of metabolites. The developed method can be used for tissue extraction setup for different indications like studies of metabolic syndrome, obesity, diabetes or cardiovascular disorders and nutrient transformation in livestock.
KeywordsMass spectrometry Tissue extraction High-throughput Quantification Complex diseases Food quality
We thank Dr. Christa Kühn from the Leibniz Institute for Farm Animal Biology (Dummerstorf, Germany) for bovine tissue samples. We thank Gabriele Zieglmeier for mouse tissue preparations, and Tamara Halex and Arsin Sabunchi for the excellent assistance in metabolomic assays. We are thankful to Dr. Gabriele Möller and Dr. Michael Urban for their advice in experimental design and tissue homogenization procedures. This study was supported in part by a grant from the German Federal Ministry of Education and Research (BMBF) to the German Center Diabetes Research (DZD e.V.) and to the research project Greifswald Approach to Individualized Medicine (GANI_MED).
- Altmaier, E., Kastenmüller, G., Römisch-Margl, W., Thorand, B., Weinberger, K. M., Adamski, J., et al. (2009). Variation in the human lipidome associated with coffee consumption as revealed by quantitative targeted metabolomics. Molecular Nutrition & Food Research, 53, 1357–1365.CrossRefGoogle Scholar
- Bogumil, R., Koal, T., Weinberger, K. M., & Dammeier, S. (2008). Massenspektrometrische Analyse von Blutplasma im Kitformat. Laborwelt, 2, 17–23.Google Scholar
- Deprez, S., Sweatman, B. C., Connor, S. C., Haselden, J. N., & Waterfield, C. J. (2002). Optimisation of collection, storage and preparation of rat plasma for 1H NMR spectroscopic analysis in toxicology studies to determine inherent variation in biochemical profiles. Journal of Pharmaceutical and Biomedical Analysis, 30, 1297–1310.PubMedCrossRefGoogle Scholar
- Fiedler, G. M., Baumann, S., Leichtle, A., Oltmann, A., Kase, J., Thiery, J., et al. (2007). Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Clinical Chemistry, 53, 421–428.PubMedCrossRefGoogle Scholar
- Folch, J., Lees, M., & Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. Journal Biological Chemistry, 226, 497–509.Google Scholar
- Freedman, D. M., Chang, S. C., Falk, R. T., Purdue, M. P., Huang, W. Y., McCarty, C. A., et al. (2008). Serum levels of vitamin D metabolites and breast cancer risk in the prostate, lung, colorectal, and ovarian cancer screening trial. Cancer Epidemiology, Biomarkers and Prevention, 17, 889–894.PubMedCrossRefGoogle Scholar
- Hettick, J. M., Green, B. J., Buskirk, A. D., Kashon, M. L., Slaven, J. E., Janotka, E., et al. (2008). Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting. Analytical Biochemistry, 380, 276–281.PubMedCrossRefGoogle Scholar
- Kratzsch, J. & Ceglarek, U. (2010). Preclinical challenges in steroid analysis of human samples. Journal of Steroid Biochemistry and Molecular Biology, in press.Google Scholar
- Kühn, C., Bellmann, O., Voigt, J., Wegner, J., Guiard, V., & Ender, K. (2002). An experimental approach for studying the genetic and physiological background of nutrient transformation in cattle with respect to nutrient secretion and accretion type. Archives Animal Breeding, 45, 317–330.Google Scholar
- U.S. Department of Health and Human Services, F. a. D. A., Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM) (2001). Guidance for Industry. Bioanalytical Method Validation.Google Scholar
- Weikard, R., Altmaier, E., Suhre, K., Weinberger, K. M., Hammon, H. M., Albrecht, E., et al. (2010). Metabolomic profiles indicate distinct physiological pathways affected by two loci with major divergent effect on Bos taurus growth and lipid deposition. Physiological Genomics, 42A, 79–88.PubMedCrossRefGoogle Scholar