High-throughput extraction and quantification method for targeted metabolomics in murine tissues

Introduction Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction. Objectives We aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics AbsoluteIDQ™ p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput. Methods We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility. Results We found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma. Conclusion We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays. Electronic supplementary material The online version of this article (10.1007/s11306-017-1312-x) contains supplementary material, which is available to authorized users.


Fig. S-1
Influence of the extraction solvent and the tissue to solvent ratio on metabolite concentrations in different mouse tissues. Page S-2

Fig. S-2
Comparison of matrix effects of murine tissue extracts to matrix effects of human plasma.
Page S-7  This figure presents the detailed data for Fig. 2 in the manuscript. Log2 fold changes were calculated for each tissue type and each metabolite class in relation to the concentrations obtained for MeOH in the respective lower tissue to solvent ratio. The fold change of the lower tissue to solvent ratio using MeOH compared to itself (fold change 0) for every tissue and metabolite class is not presented in the figure (which is in contrast to Fig. 2 in the manuscript). The figure presents the median, the 25% quartile, and the 75% quartile values for 5-6 replicates per tissue for all metabolite classes, namely for acylcarnitines (a), acyl/acyl phosphatidylcholines (PCaa, b), acyl/alkyl phosphatidylcholines (PCae, c), lysophosphatidylcholines (lysoPC, d), sphingomyelins (SM, e), amino acids (f), biogenic amines (g), and the sum of hexoses (h). Missing boxes indicate that the evaluation was not possible due to metabolite concentrations below the LOD. Abbreviations for extraction solvents are as follows: MeOH, 100% methanol; PB, 10 mM phosphate buffer pH 7.5; EtOH/PB, 85/15 (v/v) mixture of ethanol and 10 mM phosphate buffer pH 7.5. Tissue to solvent ratios are denoted as 1:X, indicating 1 mg of tissue was homogenized with X µL solvent. Metabolomics

Fig. S-2
Comparison of matrix effects of murine tissue extracts to matrix effects of human plasma.
To determine whether the matrix effects of murine tissue extracts are comparable to the matrix effects of human plasma, we calculated the ratio of the Internal Standard intensity of all tissue extracts (5-6 replicates per tissue and extraction condition) to the Internal Standard intensity of nine human reference plasma samples. Values larger than 1 indicate less ion suppression and values smaller than 1 indicate more ion suppression in tissue samples compared to human plasma. The figure presents the median, the 20% quantile, and the 80% quantile values for all metabolites measured by FIA-MS/MS (lipids, hexoses) (a), or LC-MS/MS (amino acids, biogenic amines) (b). Abbreviations for extraction solvents are as follows: MeOH, 100% methanol; PB, 10 mM phosphate buffer pH 7.5; EtOH/PB, 85/15 (v/v) mixture of ethanol and 10 mM phosphate buffer pH 7.5. Tissue to solvent ratios are denoted as 1:X, indicating 1 mg of tissue was homogenized with X µL solvent. Page S-8 Metabolomics Table S-1 List of analyzed metabolites with their abbreviations, names, metabolite classes, HMDB IDs, and MSI level of metabolite identification. The table lists metabolites covered by the analyzed MRMs, their abbreviations, names, metabolite classes, MSI level of metabolite identification, and, if available, HMDB IDs. Based on the MSI standards (Salek et al. 2013), the levels were defined as follows: Level 1 are metabolites, which were measured with LC-MS/MS (two orthogonal properties: retention time and MRM) and which had the same isotopically labeled metabolite as internal standard. Level 2 are metabolites, which were either measured with FIA-MS/MS and/or did not have an identical isotopically labeled internal standard. Chirality was not taken into account for classification.    Table S-2 Tissue metabolite concentrations at optimal extraction conditions. The table lists the mean (± standard deviation) tissue metabolite concentrations in pmol/mg tissue for each murine tissue type measured at the optimal extraction condition (summarized in Table 2 of the manuscript). The values were rounded to the first decimal. Metabolite abbreviations are explained in Table S-1. Metabolite concentrations below the LOD or higher than the ULOQ are indicated by '<LOD' or '>ULOQ', respectively. Abbreviations for extraction solvents are as follows: MeOH, 100% methanol; EtOH/PB, 85/15 (v/v) mixture of ethanol and 10 mM phosphate buffer pH 7.5. Tissue to solvent ratios are denoted as 1:X, indicating 1 mg of tissue was homogenized with X µL solvent.