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

Abstract

IntroductionGlobal 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.ObjectivesWe 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.MethodsWe 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.ResultsWe 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.ConclusionWe 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 materialThe online version of this article (10.1007/s11306-017-1312-x) contains supplementary material, which is available to authorized users.

Cholesterol metabolism promotes B‐cell positioning during immune pathogenesis of chronic obstructive pulmonary disease

“…The targeted metabolomics approach was based on LC‐ESI‐MS/MS and FIA‐ESI‐MS/MS measurements by Absolute IDQ ™ p180 Kit (BIOCRATES Life Sciences AG) which has been described in detail (Zukunft et al , ). Frozen lung tissue was homogenized and extracted as described previously (Conlon et al , ; Zukunft et al , ). Mass spectrometric analyses were done on an API 4000 triple quadrupole system (Sciex Deutschland GmbH) equipped with a 1200 Series HPLC (Agilent Technologies) and a HTC PAL auto sampler (CTC Analytics) controlled by the software Analyst 1.5.1.…”

Characterization of bulk phosphatidylcholine compositions in human plasma using side-chain resolving lipidomics

“…AbsoluteIDQ TM p150 and p180 kits have emerged as established measurement kits for quantification of phospholipids, such as PCs, in blood from thousands of participants in large epidemiological cohorts (11,12,15,16,20,(35)(36)(37)(38)(39) and in projects using a variety of human and non-human sample matrices (40)(41)(42)(43)(44). As the kits quantify PCs on the level of lipid species, each PC measure is composed of isobaric PCs (within mass resolution) with different combinations of fatty acid chain lengths and degrees of desaturation for the two residues.…”

Correlation guided Network Integration (CoNI) reveals novel genetic regulators of hepatic metabolism

“…Frozen murine liver samples were weighed and metabolites were extracted as previously described in ice-cold extraction solvent, a 85/15 (v/v) ethanol/10 mM phosphate buffer pH 7.5 mixture at a ratio of 3 μL solvent per 1 mg tissue weight [32]. The liver samples were homogenized using a Precellys24 homogenizer (PeqLab Biotechnology, Erlangen, Germany) thrice for 20s at 5,500 rpm and 4°C, with 30 s pause intervals to ensure constant temperature, followed by centrifugation at 4°C and 10,000 x g for 5 min.…”

“…The assay allows simultaneous quantification of 188 metabolites out of 10 μL tissue lysate or 20 μL cell lysate, and includes free carnitine, 39 acylcarnitines (Cx:y), 21 amino acids (19 proteinogenic + citrulline + ornithine), 21 biogenic amines, hexoses (sum of hexoses – about 90-95 % glucose), 90 glycerophospholipids (14 lysophosphatidylcholines (LPC) and 76 phosphatidylcholines (PC)), and 15 sphingolipids (SMx:y). The abbreviations Cx:y are used to describe the total number of carbons and double bonds of all chains, respectively [32, 34]. For the LC-part, compound identification and quantification were based on scheduled multiple reaction monitoring measurements (sMRM).…”