Liver (~100 mg) was lysed in 1 mL of phosphate-buffered saline with a bead mill homogenizer (VWR, Radnor, PA). Lipids were extracted using Folch’s method.(25 (link)) The organic phase of each sample, normalized by tissue weight, was separated using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry.(26 (link)) See Supporting Methods for details. All data were analyzed using the LipidSearch version 4.1 SP (Thermo Fisher Scientific), and all identified species (grade A, B, and C quality) were validated using R studio (RStudio Team [2015]; RStudio: Integrated Development for R; RStudio, Inc., Boston, MA; www.rstudio.com ). The data were manually curated after computational analysis. Data are displayed as median fold change compared to control chow-fed animals, scaled linearly.
Lipidsearch version 4.1 sp
Manufactured by Thermo Fisher Scientific
LipidSearch version 4.1 SP is a software application developed by Thermo Fisher Scientific for the analysis and identification of lipids in complex biological samples. The software utilizes high-resolution mass spectrometry data to perform lipid profiling and quantification.
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Lab products found in correlation
2 protocols using lipidsearch version 4.1 sp
1
Liver Lipid Extraction and Profiling
2
Comprehensive Liver Lipid Profiling
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Liver (∼100 mg) was homogenized in 1 ml of PBS using a Bead Mill Homogenizer (VWR). Lipids were extracted, according to the Folch method (31 (link)). Lysis volume was normalized to starting tissue material. The organic fraction containing extracted lipids was subjected to liquid chromatography/tandem mass spectrometry analysis as described in (28 (link)). Mass spectrometry data analysis was performed using LipidSearch version 4.1 SP (Thermo Fisher Scientific). The results were exported to R-Studio where quality control was performed using pairwise correlations between replicates, a principal component analysis comparing sample groups, as well as retention time plot analysis to verify elution clustering within lipid classes. All identified lipids were included for subsequent analyses if they fulfilled the following LipidSearch-based criteria: (1) reject equal to zero, (2) main grade A or main grade B and a p value of <0.01 for at least three replicates, and (3) no missing values across all samples. Statistical significance was calculated using a Student t test followed by a Holm-Sidak test to correct for multiple comparisons.
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