DOI: 10.5061/dryad.vx0k6djvm
title: Lipidomic datasets for: Transmembrane protein 135 regulates lipid homeostasis through its role in peroxisomal DHA metabolism

description: Transmembrane protein 135 (TMEM135) is thought to participate in the
 cellular response to increased intracellular lipids yet no defined
 molecular function for TMEM135 in lipid metabolism has been identified. In
 this study, we performed a lipid analysis of tissues
 from Tmem135 mutant mice and found striking reductions
 of docosahexaenoic acid (DHA) across all Tmem135 mutant
 tissues, indicating a role of TMEM135 in the production of DHA. Since all
 enzymes required for DHA synthesis remain intact
 in Tmem135 mutant mice, we hypothesized that TMEM135 is
 involved in the export of DHA from peroxisomes.
 The Tmem135 mutation likely leads to the retention of
 DHA in peroxisomes, causing DHA to be degraded within peroxisomes by their
 beta-oxidation machinery. This may lead to generation or alteration of
 ligands required for the activation of peroxisome proliferator-activated
 receptor a (PPARa) signaling, which in turn could result in increased
 peroxisomal number and beta-oxidation enzymes observed
 in Tmem135 mutant mice. We confirmed this effect of
 PPARa signaling by detecting decreased peroxisomes and their proteins upon
 genetic ablation of Ppara in Tmem135 mutant
 mice. Using Tmem135 mutant mice, we also validated the protective
 effect of increased peroxisomes and peroxisomal beta-oxidation on the
 metabolic disease phenotypes of leptin mutant mice which has been observed
 in previous studies. Thus, we conclude that TMEM135 has a role in lipid
 homeostasis through its function in peroxisomes.
description: <strong>Dataset was collected by the following
 methods:</strong> <strong>Sample
 preparation for lipidomics analysis. </strong>Livers, retinas
 (neural retinas and eyecups), hearts, and plasmas were collected and
 stored at -80°C prior to lipid extraction. Prior to extraction, all
 solutions were pre-chilled on ice. Samples were transported and cut on a
 dry ice-cooled stainless steel plate and weighed on a Mettler (XSR205)
 analytical balance to the nearest hundredth of a milligram. This weight
 would be used to normalize samples for analysis. Fifty microliter aliquots
 of plasma from each mouse were used in this study. No normalization
 occurred with the plasma samples since the same volume for each sample was
 used in this study. Tissues were then placed into Qiagen PowerBead tubes
 (P/N 13113-50) for homogenization and extraction. Lipids were extracted in
 a solution of 250 µL PBS, 225 µL methanol containing internal standards
 (Avanti SPLASH LipidoMix (Lot#3307-07) at 10 µL per sample) and 750 µL
 MTBE (methyl tert-butyl ether). The samples were homogenized in three
 30-second cycles alternated with a 5-minute rest on ice using a Qiagen
 TissueLyzer II operated at 30 Hz. The final rest on ice was 15 minutes.
 After centrifugation at 16,000 g for 5 minutes at 4°C, 500 µL of the upper
 phase was collected in 1.5 ml centrifuge tubes and evaporated to dryness
 in a Savant speedvac concentrator. Lipid samples were reconstituted in 150
 µL of isopropanol.  A process blank was prepared in parallel with the
 tissue samples during extraction and analyzed concurrently with the
 samples. A pooled sample, used for lipid identification and quality
 control, was prepared by taking equal volumes from each sample of a given
 tissue after final resuspension in IPA.
 <strong>LC-MS methods for lipidomics. </strong>Sample
 analysis was done at different dilution factors for different tissues and
 ionization polarities. For each injection samples were diluted in
 isopropanol by adding a given sample volume to isopropanol in an LC-MS
 vial with deactivated glass insert (Agilent P/N 5182-0554 and 5183-2086),
 vortexed to mix, and spun at low speed to collect the liquid prior to
 placing in the autosampler for analysis. Lipid extracts were separated on
 a Waters Acquity UPLC BEH C18 1.7 µm 2.1 x 100 mm column coupled in tandem
 with a Waters Acquity UPLC BEH C18 1.7 µm VanGaurd pre-column 2.1x5mm and
 maintained at 50°C. These columns in turn were connected to an Agilent HiP
 1290 Multisampler, Agilent 1290 Infinity II binary pump, and column
 compartment connected to an Agilent 6546 Accurate Mass Q-TOF dual ESI mass
 spectrometer. For positive ion mode, the source gas temperature was set to
 250°C, with a gas flow of 12 L/min and a nebulizer pressure of 35 psig.
 VCap voltage was set at 4000 V, fragmentor at 145 V, skimmer at 45 V and
 Octopole RF peak at 750 V. For negative ion mode, the source gas
 temperature was set to 350°C, with a drying gas flow of 12 L/min and a
 nebulizer pressure of 25 psig. VCap voltage is set at 5000 V, fragmentor
 at 200 V, skimmer at 45 V and Octopole RF peak at 750 V. Reference masses
 in positive mode (<em>m/z </em>121.0509 and 922.0098) and
 negative mode (<em>m/z</em> 966.0007, and 112.9856) were
 delivered to the second emitter in the dual ESI source by isocratic pump
 at 15 uL/min. Samples were analyzed in a randomized order in both positive
 and negative ionization modes in separate experiments acquiring with the
 scan range m/z 100–1500. Mobile phase A consisted of acetonitrile:water
 (60:40 v/v) containing 10 mM ammonium formate and 0.1% formic acid, and
 mobile phase B consists of isopropanol: acetonitrile:water (90:9:1 v/v)
 containing 10 mM ammonium formate and 0.1% formic acid. The chromatography
 gradient for both positive and negative modes started at 15% mobile phase
 B then increases to 30% B over 2.4 min, then increased to 48% B from
 2.4–3.0 min, then increased to 82% B from 3–13.2 min, then increased to
 99% B from 13.2–13.8 min where it was held until 15.4 min and then
 returned to the initial conditions and equilibrated for 4 min. Flow was
 0.5 mL/min throughout the gradient.  Injection volumes were 2 µL for
 positive mode and 5 µL for negative mode MS1 acquisitions, and 4 µL for
 positive mode and 7 µL for negative mode MS/MS acquisitions. Tandem mass
 spectrometry (MS/MS) was conducted using the same LC gradient as above
 with isolation width set to “narrow” (~1.3 m/z) and collision energy of 25
 V. MS/MS data was collected on the pooled sample per tissue in iterative
 mode, in which the sample is analyzed 5 times with different precursors
 selected upon each injection. This permits access to lower-abundance lipid
 species in a background of highly abundant lipids.  
 <strong>Dataset has been processed by the following
 methods:</strong> <strong>Data
 Analysis.</strong> Pooled MS/MS and individual sample MS data were
 analyzed using a combination of Agilent and web-based applications. Lipid
 identification was achieved from the pooled MS/MS data via Lipid Annotator
 (Agilent). This software utilizes accurate mass m/z values for the intact
 lipid and experimental and theoretical fragment ion m/z values to assign
 lipid class and alkyl chain identities where possible. In some cases,
 specific alkyl chain identities cannot be assigned, but the sum
 composition of those alkyl chains (i.e. carbon number and degree of
 unsaturation) can be determined and that is reported instead. The output
 from Lipid Annotator is a database of lipid species with m/z values and
 retention times for each. This process was performed independently for
 positive and negative ion modes. Quantitation of lipids in individual
 samples used MS data alone. In this analysis, data for each sample was
 collected separately. The abundance of each lipid in each sample was
 tabulated using the Profinder application (Agilent). In this analysis, the
 database of lipid identity, m/z values (for protonated, deprotonated,
 ammonium or sodium adducted, or formate adducted, depending on ionization
 mode) and retention time was used to extract an ion chromatogram for each
 lipid species. These chromatograms were then integrated to produce an area
 for that lipid in each sample.  These abundance values were then exported
 as a .csv file for statistical analysis using the web-based tool
 MetaboAnalyst 5.0. This software was used for principal component analysis
 and statistical testing. The datasets provided in this depository are the
 .csv files for the MetaboAnalyst 5.0 program.
description: The programs required to open the data files include Microsoft
 Excel and MetaboAnalyst 5.0 (https://www.metaboanalyst.ca).