The lipidomic profile was determined in an external laboratory, the Centre for Omic Sciences (COS) in Reus, Spain. Whole brain samples were homogenized, and 5 mg of the tissue was extracted with chloroform:methanol. The lower phase was recovered after centrifuging with water and NaCl (0.9%), and reconstituted with methanol:methyl-tert-butyl ether. Analysis was performed by UHPLC-qTOF (model 6550 of Agilent, USA) in positive electrospray ionization mode. Chromatographic gradient elution with a ternary mobile phase containing water, methanol and 2-propanol with 10 mM ammonium formate and 0.1% formic acid was performed using a C18 column (Kinetex EVO C18 Column, 2.6 μm, 2.1 mm × 100 mm) as a stationary phase, allowing the sequential elution of the more hydrophobic lipids. The lipid species were identified by matching their accurate mass and tandem mass spectrum to Metlin-PCDL from Agilent and by matching the chromatographic behavior of pure standards for each family of lipids. Then, lipids were semi-quantified depending on their family similarity by internal standard calibration curves using pure chemical standards. Finally, concentration was calculated by normalization with the original tissue weight for each brain sample.
Metlin pcdl
Manufactured by Agilent Technologies
Metlin-PCDL is a comprehensive database of small molecule compounds provided by Agilent Technologies. It contains detailed information on the chemical structures, physical properties, and mass spectrometric data for a large number of chemical compounds. The database is designed to facilitate the identification and quantification of small molecules in complex samples using mass spectrometry techniques.
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Lab products found in correlation
4 protocols using metlin pcdl
1
Lipidomic Profiling of Whole Brain
2
Lipid Identification and Characterization
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Lipid extraction procedure can be found in the Supplementary Methods . The identification of lipid species was performed using the Agilent MassHunter Profinder B.08 software. First, a feature extraction deconvolution was made; accurate mass and tandem mass spectrum, when available, was then matched to Metlin-PCDL (2017) from Agilent containing more than 40 000 metabolites and lipids, allowing a mass error of 20 ppm and a score higher than 80 for isotopic distribution. To ensure the tentative characterization, the chromatographic behaviour of pure standards for each family and corroboration with the Lipid Maps database (www.lipidmaps.org ) was used to ensure their putative identification. Afterwards, matched entities were selected to perform a targeted MS/MS acquisition on the LC-QTOF-MS instrument to corroborate the identification. Lipid species were then semiquantified in terms of internal standard response ratio using one internal standard for each lipid family.
3
Metabolite Analysis in Brain and Liver
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The analysis of metabolites in extracts from brain and liver was performed according to the Agilent METLIN/PCDL method using a 1290 infinity UHPLC coupled to a 6550 ESI-QTOF, as previously described [36 (link)].
4
Lipid Identification and Quantification
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The MS data were processed using both Agilent MassHunter Qualitative and Quantitative Analysis B.07 software. The identification of lipid species was performed by matching their accurate mass and tandem mass spectrum, when available, to Metlin-PCDL from Agilent containing more than 40,000 metabolites and lipids. LipidCreator workbench was also used for targeted exact mass list generation [56 (link)]. In addition, chromatographic behaviour of pure standards for each family and bibliographic information was used to ensure their putative identification. After putative identification of lipids, these were quantified in terms of internal standard response ratio using one internal standard for each lipid family by using Agilent Mass Hunter Quantitative software (B.07) to create a refined matrix of quantitative data for statistical purposes.
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