Amino acids and biogenic amines were quantified based on isotopically labeled internal standards and 7-point calibration curves using AB Sciex Analyst®® version 1.7.2 software. Other metabolites, such as acylcarnitines, lysoPCs, PCs, SMs and hexoses, were semi-quantified using 14 internal standards in MetIDQ™ software version 8.7.1(Biocrates Life Sciences). Data quality was evaluated by checking the accuracy and reproducibility of QC samples included in the p180 kit. Finally, the concentrations of metabolites were reported in µM. For further statistical analyses, metabolites were included only when the concentrations of metabolites were above the limit of detection (LOD) in more than 50% of samples.
Metidq
Manufactured by Biocrates
Sourced in Austria
MetIDQ is a comprehensive software solution for the acquisition, processing, and analysis of metabolomics data. It supports multiple analytical platforms, including mass spectrometry and nuclear magnetic resonance spectroscopy. MetIDQ facilitates the identification and quantification of a wide range of metabolites, enabling comprehensive metabolic profiling.
Automatically generated - may contain errors
Lab products found in correlation
Surveyor hplc system, by Thermo Fisher Scientific (6 mentions)
4000 qtrap, by AB Sciex (3 mentions)
Tsq vantage emr, by Thermo Fisher Scientific (2 mentions)
Zorbax eclipse xdb c18, by Agilent Technologies (2 mentions)
Absoluteidq p180 kit, by Biocrates (2 mentions)
Mxp quant 500 kit, by Biocrates (2 mentions)
Qtrap 6500 system, by AB Sciex (1 mentions)
Sciex analyst, by AB Sciex (1 mentions)
5500 qtrap instrument, by AB Sciex (1 mentions)
Metaboindicator, by Biocrates (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Absoluteidq 180 kit, by Biocrates (1 mentions)
Absolute idq p400 hr kit, by Biocrates (1 mentions)
15 protocols using metidq
1
Targeted Metabolite Profiling in Samples
2
Targeted Lipidomics by ESI-MS/MS
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Targeted lipidomics was performed in collaboration with Biocrates (Innsbruck, Austria). The different bioactive lipids (ie, 12-HETE, 6-keto-PGF1a, 8-iso-PGF2a, 9-HODE, 13-HODE, 15-HETE, AA, DHA, LTB4, PGD2, PGE2, PGF2a and TXB2) were quantitatively analysed using a high-throughput flow injection electrospray ionisation mass spectrometry (ESI-MS/MS) screening method. Multiple reaction monitoring (MRM) detection in positive and negative modes was performed using an AB SCIEX 4000 QTrap tandem mass spectrometry instrument (AB SCIEX, Darmstadt, Germany). The sample was prepared in a 20 µL volume, followed by an MeOH/CHCl3-liquid/liquid-extraction protocol. Internal standards were used to compensate for matrix effects, and external standards were used for multipoint calibration. The quantitative analysis was performed with Biocrates in-house software MetIDQ enabling isotopic correction.
3
Targeted Metabolomics Profiling of Serum
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Serum samples (10 µL) were analyzed by a targeted quantitative approach using a combined direct flow injection and liquid chromatography (LC) tandem mass spectrometry (MS/MS) assay using the Absolute IDQ p180 kit (Biocrates Life Sciences AG) according to the manufacturer’s protocol. This strategy hypothetically allows simultaneous quantification of 185 metabolites: 40 amino acids and biogenic amines, 40 acylcarnitines, 90 glycerophospholipids, and 15 sphingomyelins. The method combines derivatization and extraction of analytes with the selective mass-spectrometric detection using multiple reaction monitoring and integrated isotope-labeled internal standards absolute quantification. Mass spectrometry analyses were carried out on a TSQ Vantage EMR (Thermo SCIENTIFIC) equipped with a Surveyor HPLC system (Thermo SCIENTIFIC) using an Agilent Zorbax Eclipse XDB-C18 (3.5 μm) 3.0 × 100 mm column and controlled by Xcalibur 2.1. software. The acquired data were processed using Xcalibur 2.1. and MetIDQ (Biocrates Life Sciences AG) software. Data normalization was performed according to the kit manufacturer’s protocol based on the standard serum sample. Concentrations of all metabolites were calculated in μM.
4
Quantitative Plasma Metabolomics via ESI-LC-MS/MS
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A targeted metabolomics approach based on electrospray ionization–liquid chromatography–mass spectrometry (ESI–LC–MS/MS) and MS/MS measurements was performed using the AbsoluteIDQ™ p180 Kit (Biocrates Life Sciences, Innsbruck, Austria). The assay allows the simultaneous quantification of a total of 188 metabolites from 10 µL plasma samples. This study focuses on the quantification of carnitine, as well as of the C2, C3, C3-DC/C4-OH, C4, C5, C10, C14:1, C14:2, C16, C18, C18:1, and C18:2 acylcarnitines. The analyses were carried out on a 4000 QTRAP® System (Sciex Deutschland, Darmstadt, Germany) and a Thermo Scientific™ TSQ™ (ThermoFisher Scientific, Waltham, MA, USA). For the evaluation of the metabolites’ concentrations, internal standards served as a reference. The MetIDQ™ (Biocrates Life Sciences, Innsbruck, Austria) software was used for the processing and the technical validation of the metabolite data.
5
Comprehensive Metabolite Profiling by LC-MS
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Amino acids and biogenic amines were acquired by liquid chromatography (10 μl injection volume) and acylcarnitines, glycerophospholipids, sphingolipids and total hexoses by flow injection analysis (20 μl injection volume), both coupled with tandem mass spectrometry in positive and negative electrospray ionization modes (S1 Table ). All the analyses were performed on a QTRAP 6500 System (AB Sciex, Framingham, MA).
To avoid run-order effects, serum samples were analysed in a randomized batch format. Quality controls were analysed throughout the whole run to control the stability and performance of the system and evaluate the quality of the acquired data [11 (link)]. Metabolites were quantified by multiple reaction monitoring, by reference to multipoint calibration curves and/or in combination with the use of stable isotope-labelled and other internal standards, to compensate for matrix effects [12 ].
Isotopic correction was performed with the software MetIDQ™ (Biocrates Life Sciences AG). The limits of detection (LOD) and lower (LLOQ) and upper limits of quantification were experimentally determined.
To avoid run-order effects, serum samples were analysed in a randomized batch format. Quality controls were analysed throughout the whole run to control the stability and performance of the system and evaluate the quality of the acquired data [11 (link)]. Metabolites were quantified by multiple reaction monitoring, by reference to multipoint calibration curves and/or in combination with the use of stable isotope-labelled and other internal standards, to compensate for matrix effects [12 ].
Isotopic correction was performed with the software MetIDQ™ (Biocrates Life Sciences AG). The limits of detection (LOD) and lower (LLOQ) and upper limits of quantification were experimentally determined.
6
Targeted Plasma Metabolomics by LC-MS/MS
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Tandem mass spectrometry was performed for targeted metabolomics of plasma samples using Biocrates MxP® Quant 500 Kit (Biocrates, Innsbruck, Austria) at the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM). FIA-MS/MS was utilized to measure lipids using a 5500 QTRAP® instrument with an ESI source (AB Sciex, Darmstadt, Germany). LC-MS/MS utilizing the same 5500 QTRAP® instrument was used for measuring small molecules as described previously (17 (link)). Appropriate MS software (Sciex Analyst®) was used to quantify data that was then imported into Biocrates MetIDQ™ software for calculation of analyte concentrations, assessment and compilation of data.
7
Comprehensive Lipidomics Analysis of Plasma
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Lipidomics analysis was carried out by BIOCRATES Life Sciences AG (Biocrates Life sciences AG, Innsbruck, Austria) on lithium heparin plasma samples. The biologically most abundant members of (lyso-) glycerophospholipids, i.e. (lyso-) glycerolphosphocholines, −ethanolamines, −serines, −glycerols, as well as sphingolipids, i.e. sphingomyelins, ceramides, dihydroceramides, and 2-hydroxyacyl ceramides, were quantitatively analysed by a high throughput flow injection ESI-MS/MS method as previously described [15 (link), 53 (link)]. The quantitative data analysis was performed with Biocrates in-house software MetIDQ™ enabling isotopic correction and basic statistical analysis. To deal with data at or below the limit of detection only lipids which had ≥ 70 % of subjects above the limit of detection were included in this analysis [54 (link)]. Therefore from the 325 lipids that were measured, 240 lipids were included for data analysis. The median intra-day CV was < 8 % taking into account all measured lipids. The median inter-day CV was 16 % taking into account all lipids.
8
Lipid Profiling in Snap-frozen Tissues
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Snap frozen kidneys, hearts, brains and skeletal muscles were sent to Biocrates Inc., Innsbruck, for lipid mass spectrometry. The biologically most abundant members of (lyso-) glycerophospholipids, i.e. (lyso-) glycerophospho-cholines, -ethanolamines, -serines, -glycerols, and sphingolipids, i.e. sphingomyelins, ceramides, dihydroceramides, and 2-hydroxyacyl ceramides were quantitatively analyzed by a high throughput flow injection ESI-MS/MS screening method. The MRM detection in positive and negative mode was performed using a 4000 QTrap® tandem mass spectrometry instrument (AB SCIEX). The sample preparation of 20μL sample volume followed a MeOH/CHCl3 -liquid/liquid-extraction protocol. Besides five internal standards to compensate for matrix effects, 43 external standards were used for a multipoint calibration. The quantitative data analysis was performed with Biocrates MetIDQ™ enabling isotopic correction and basic statistical analysis.
Clustering analysis was done using Perseus software with euclidian distances. JMP (SAS, Böblingen) was used for quantitative analysis (3way-ANOVA) and visualization of results.
Clustering analysis was done using Perseus software with euclidian distances. JMP (SAS, Böblingen) was used for quantitative analysis (3way-ANOVA) and visualization of results.
9
Targeted Metabolomic Analysis of Plasma
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Targeted metabolomic analysis in plasma collected at 8 weeks was performed using the commercially available MxP® Quant 500 kit (Biocrates Life Sciences, Innsbruck, Austria), following the manufacturer’s instructions provided. Added to a 96-well plate were 10 μL of plasma, calibrators (7 levels), and quality controls (3 levels). The MxP® Quant 500 kit can potentially identify 630 metabolites across 23 classes of compounds by LC-MS/MS. Peak identification was accomplished using Analyst 1.7.2 (SCIEX, Framingham, MA, USA) and multiple reaction monitoring. MS data were uploaded to MetIDQ (Biocrates, Innsbruck, Austria) for analyte quantitation. MetaboIndicatorTM (biocrates life sciences, Innsbruck, Austria) was used to calculate 232 metabolic indicators, based on the sums and/or ratios of metabolites.
10
Quantification of Metabolic Biomarkers
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Amino acids and biogenic amines were quantified based on isotopically labelled internal standards and 7-point calibration curves in Analyst version 1.7.2 software. Other metabolites such as acylcarnitines, LPCs, PCs, SMs and hexose were measured semi-quantitatively by using 14 internal standards within the MetIDQ™ software (Biocrates). Metabolite concentrations are reported in µM.
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