Supporting Information - PNAS dodecylmonoalkylglycerol ether (C12:0 MAGE) were pur-chased from Sigma-Aldrich. Monopentadecanoin (C15:0 MAG) andmonoheptadecanoin(C17:0MAG)werepurchasedfromNu-

Supporting InformationBlankman et al. 10.1073/pnas.1217121110SI Materials and MethodsMaterials. We purchased 2-arachidonoylglycerol (2-AG) fromCayman Chemicals; 2-oleoylglycerol, pentadecanoic acid (PDA),and dodecylmonoalkylglycerol ether (C12:0 MAGE) were pur-chased from Sigma-Aldrich. Monopentadecanoin (C15:0 MAG)andmonoheptadecanoin (C17:0MAG) were purchased fromNu-Chek-Prep. Phospholipids and lysophospholipids were purchasedfrom Avanti Polar Lipids. Fluorophosphonate (FP)-rhodamine(1) and JZL184 (2) were synthesized as described previously.

Generation of ABHD12−/− Mice. The α/β-hydrolase domain-con-taining (ABHD)12-targeting construct was generated by ampli-fying 3.4- and 4.4-kb regions of the Abhd12 gene adjacent to thecatalytic exon 8 from a BAC clone containing the Abhd12 locus(clone ID RP23-193L22 from BACPAC Resources Center atChildren’s Hospital Oakland Research Institute) and subcloningthese homologous arms into the SacII/BamHI and XhoI/HindIIIsites of the pKO-NTKV vector. The targeting construct wasdesigned to replace Abhd12 exons 8–10 with a neomycin selec-tion cassette upon homologous recombination. Following elec-troporation of the targeting construct in C57BL/6 Bruce 4-derivedmurine embryonic stem (ES) cells, 300 integrated ES cell cloneswere obtained and screened for homologous recombination bySouthern blot analysis with external probes located 50 and 30 of thetargeted region. These probes gave differential band sizes in WT(10.4 kb) or targeted (50 probe, 4.8 kb; 30 probe, 5.7 kb) genomicDNA digested with EcoRI. One homologous recombinant, 96, wasidentified, expanded and injected into albino C57BL/6 blastocysts.Blastocyst implantation into pseudopregnant albino C57BL/6 fe-males generated nine chimeric males. Three of the chimeras pro-duced germ-line transmission of the targeted mutation, which wasconfirmed by Southern analysis. PCR genotyping of genomic tailDNA was performed using the primers 50-CAGTGCTGGCCT-GTCAGTCG-30, 5-GGTGCCCAGTGAATGGCC-30, and 50-TA-AAGCGCATGCTCCAGACTGCC-30, which amplify a 467-bpproduct from the WT allele and an 295-bp product from thetargeted allele. All mice used in this study were generated frombreeding ABHD12+/− mice and had ad libitum access to waterand food.

Biochemical Studies. Preparation of mouse brain proteomes.Mice wereanesthetized with isoflurane and killed by decapitation. Brainswere harvested, sectioned into hemispheres, immediately flashfrozen in liquid nitrogen, and frozen at −80 °C until use. One halfbrain was Dounce-homogenized in PBS (pH 7.5), sonicated, andcentrifuged at slow speed (1,400 × g for 10 min at 4 °C) to re-move debris. The supernatant was centrifuged at high speed(100,000 × g for 45 min at 4 °C), and this supernatant was savedas the soluble proteome. The pellet was washed and resuspendedin PBS, sonicated, and saved as the membrane proteome. Thetotal protein concentration of each proteome was determinedusing the Bio-Rad Dc Protein Assay kit. Aliquots of the pro-teomes were stored at −80 °C until use.Activity-based protein profiling analysis. Brain membrane proteomes(50 μg in 50 μL of PBS buffer) were prepared from 6-wk-oldABHD12−/−, ABHD12+/−, and ABHD12+/+ littermates and pre-treated with 5 μM JZL184 or DMSO vehicle for 30 min at 25 °C,followed by incubationwith 2 μMFP-rhodamine (1) for 1 h at 25 °C.Reactions were quenched with 2× SDS/PAGE loading buffer (re-ducing), separated by SDS/PAGE [10% (wt/vol) acrylamide] andvisualized in-gel with a FMBio IIe flatbed fluorescence scanner(Hitachi). Rhodamine fluorescence is shown in gray scale.

RT-PCR. Total RNA was isolated from brain tissue of 6-wk-oldABHD12−/− and ABHD12+/+ mice using TRIzol (Invitrogen).First-strand cDNA was synthesized using the SuperScript IIIReverse Transcriptase kit (Invitrogen) according to the manu-facturer’s protocol. PCR amplification (25 cycles) of a 259 bpfragment of the ABHD12 cDNA was performed with primers 50-CTCAGTAGGAACACCATCGGAGC-30 and 50-GCCAGGG-AAGTATCGGTATATCACTG-3. Amplification of a 245-bpGAPDH product was performed as a control with primers 50-GGTGAAGGTCGGTGTGAACGG-30 and 50-CCCATTTGA-TGTTAGTGGGGTCTCG-30. Both sets of primers were de-signed to span a >2-kb region of genomic DNA.Untargeted liquid chromatography–mass spectrometry proteomic analysis.Mouse brain membrane proteomes were prepared from 6-mo-oldABHD12−/− and ABHD12+/+ mice (n = 3 per genotype). Mem-brane proteins (0.375 mg of total protein) were precipitated with1:4 (vol/vol) CHCl3:MeOH and denatured with 25 mM ammo-nium bicarbonate in 6 M urea. Samples were reduced with10 mM DTT, alkylated with 40 mM iodoacetamide, and dilutedto 2 M urea with 25 mM ammonium bicarbonate. Digestion withtrypsin (0.5 μg/μL) was performed overnight at 37 °C in thepresence of 1 mM CaCl2. The tryptic peptide samples wereacidified with 5% (vol/vol) formic acid, and aliquots were frozenat −80 °C until use.Multidimensional protein identification technology (MudPIT)

analysis was performed as described previously (3) on an LTQOrbitrap Velos mass spectrometer (ThermoFinnigan) coupled toan Agilent 1200 series HPLC (50 μg of protein; 11-step gradi-ent). MS spectra were acquired in profile mode, with a massrange of 400–1,800 in the Orbitrap analyzer with resolution set at30,000, followed by 30 MS/MS scans in the ion trap. Dynamicexclusion was enabled with a repeat count of 1, a repeat durationof 20 s, exclusion duration 20 s, and an exclusion list size of 300.All tandem mass spectra were collected using a normalizedcollision energy of 35%, an isolation window of 2 Da, and anactivation time of 10 ms. One microscan was applied for all ex-periments. Spray voltage was set to 2.50 kV, and the flow ratethrough the column was 0.20 μL/min.RAW files were generated from the mass spectra using

XCalibur version 1.4, and MS/MS spectra data extracted usingRAW Xtractor (Version 1.9.1), which is publicly available(http://fields.scripps.edu/?q=content/download). The tandemMS data were searched against the mouse International ProteinIndex (IPI) database using the ProLuCID search algorithm (4),allowing for modification of M with the crosslinking agent(15.9949), static modification of cysteine residues (57.02146 Da,because of alkylation), half-tryptic enzyme specificity, and a masstolerance set to 50 ppm for precursor mass and ±0.6 Da forproduct ion masses. The resulting MS/MS spectra matches wereassembled and filtered using DTASelect2 (Version 2.0.27). Thevalidity of peptide/spectrum matches was assessed using DTA-Select2 (Version 2.0.27) and two SEQUEST-defined parame-ters: the cross-correlation score (XCorr) and the normalizeddifference in cross-correlation scores (DeltaCN). Peptides withXCorr scores greater than 1.8 (+1), 2.5 (+2), or 3.5 (+3), andDeltaCN scores greater than 0.08 were included in the spectralcounting analysis.Lipid substrate hydrolysis assays. The 2-AG hydrolysis activity ofbrain membrane homogenates fromABHD12−/− and ABHD12+/+

mice (2 mo old; n = 4 per genotype) plus 1 μM JZL184 or vehicle(DMSO) was measured as described previously (5). Assessing theactivity of brain membrane homogenates against C18:1 mono-

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acylglycerol (MAG), C18:1 lysophosphatidylserine (LPS), C18:1lysophosphatidylinositol (LPI), C18:1 lysophosphatidylglycerol(LPG), C18:1 lysophosphatidylcholine (LPC), C18:1 lysophos-phatidic acid (LPA), and C18:1/18:1 bis(monoacylglcerol)phos-phate (BMP) was performed similarly. Mouse brain membraneproteomes were prepared from 6-mo-old ABHD12−/− andABHD12+/+ littermates (n = 3 per genotype). Activity assayswere performed with 20 μg of brain membrane homogenate di-luted in PBS (100 μL total volume) incubated with 100 μM lipidsubstrate. After 30 min at 25 °C, the reactions were quenchedwith 2:1 (vol/vol) CHCl3:MeOH (350 μL), and 0.5 nmol PDAwas added as an internal standard. The reaction vials were vor-texed to mix and centrifuged for 5 min at 1,400 × g to separatephases. A portion of the lower organic phase (30 μL) was in-jected onto an Agilent 6520 series quadrupole–time-of-flight(QTOF) MS. Chromatography was performed on a 50 × 4.60mm 5-μmGemini C18 column (Phenomenex) coupled to a guardcolumn (Gemini; C18; 4 × 3.0 mm; Phenomenex SecurityGuardcartridge). The LC method consisted of 0.1 mL/min of 100%buffer A [95:5 (vol/vol) H2O:MeOH plus 0.1% (vol/vol) am-monium hydroxide] for 1.5 min, 0.5 mL/min linear gradient to100% buffer B [65:35:5 (vol/vol) iPrOH:MeOH:H2O plus 0.1%(vol/vol) ammonium hydroxide] over 5 min, 0.5 mL/min 100%buffer B for 5.5 min, and equilibration with 0.5 mL/min 100%buffer A for 3 min (15 min total run time). MS analysis wasperformed in negative scanning mode with an electrospray ion-ization (ESI) source. The capillary voltage was 4.0 kV, thefragmentor voltage was 100 V, the drying gas temperature was350 °C, the drying gas flow rate was 11 mL/min, and the nebu-lizer pressure was 45 psi. Product (oleic acid for lysophospholi-pids and C18:1 LPG for BMP) release was quantified bymeasuring the area under the peak in comparison with the PDAinternal standard and corrected for the nonenzymatically formedproduct present in heat inactivated (10 min at 90 °C) controlreactions. Data are presented as the average of three biologicalreplicates, error bars represent the SEM, and statistical signifi-cance was calculated using the Student’s t test.Recombinant ABHD12 activity against a panel of lipid sub-

strates was assessed by a liquid chromatography–mass spec-trometry (LC-MS) method essentially as described previously(6). HEK293T cells were grown to ∼70% confluence in 10 cmdishes in Dulbecco’s modified Eagle medium (DMEM) supple-mented with L-glutamine and 10% (vol/vol) FBS at 37 °C and 5%(vol/vol) CO2. Cells were transiently transfected with 6 μg of themurine ABHD12 cDNA in pSPORT6-CMV (OpenBioSystems)or empty vector control (“mock”) using the FUGENE 6 (RocheApplied Science) transfection reagent according to the manu-facturer’s protocols. After 48 h, the cells were washed twice withPBS, collected by scraping, resuspended in 500 μL of PBS, andlysed by sonication. The lysates were centrifuged (100,000 × g for45 min at 4 °C), and the pellet was washed, resuspended in PBS,sonicated, and saved as the membrane homogenate. Proteinconcentrations were determined using the Bio-Rad Dc proteinassay, and aliquots of the homogenates were stored at −80 °Cuntil use. Successful overexpression of active ABHD12 proteinwas confirmed by activity-based protein profiling (ABPP) anal-ysis as described above. ABHD12-expressing or mock cellmembrane homogenates (5 μg) in PBS (100 μL total volume)were incubated at 25 °C with the lipid substrates listed in TableS7. After 30 min, the reactions were quenched by the addition of350 μL of 2:1 vol/vol CHCl3:MeOH, doped with 0.5 nmol of in-ternal standards (Table S7), vortexed, then centrifuged (1,400 × g,3 min) to separate the phases. A portion of the organic phase (20μL) was injected onto an Agilent 6520 QTOF MS. LC separationand MS parameters were identical to those described above,except that the reaction products for dioleoylglycerol (DOG) andphosphatidylcholine (PC) were measured in positive-ion modewith mobile phase buffers containing 0.1% (vol/vol) formic acid.

The fatty acid or lysophospholipid hydrolysis products werequantified by measuring the area under the peak in comparisonwith the internal standard and corrected for the backgroundproduct present in reactions performed with heat inactivated (10min at 90 °C) protein homogenates. Data are presented as themean of three replicates ± SEM. Statistical analysis was performedby Student’s t test.

Behavioral Analysis. All behavioral testing, except for the tetradtests for cannabimimetic activity, was performed in The ScrippsResearch Institute mouse behavior assessment core facility. Micewere group-housed in a temperature-controlled room in whichthe lights were on a 12-h light/dark cycle (lights off at 0600 hours),and behavior was assessed during the dark (active) phase. Foodand water were available ad libitum.A cohort of ABHD12−/−, ABHD12+/−, and ABHD12+/+ lit-

termates (n = 8–9 per genotype; mixed sex) were tested for lo-comotor activity in an open-field, startle responses to auditorystimuli, rotarod performance, and hanging wire performance at5–6, 11–12, and 17–18 mo of age. A second cohort (n = 8–10 pergenotype; mixed sex) was tested at 12 and 18 mo of age. Theresults obtained at 11–12 and 17–18 mo old from both cohortswere combined.Locomotor activity. Locomotor activity was measured using anopen-field activity monitor in a square Plexiglas arena (software,hardware, and protocols from Med Associates) (7). The totalnumber of line crosses were recorded during a 10-min testingsession. Data are presented as average values ± SEM. Statisticalanalysis was performed by ANOVA and Fisher’s protected least-significant difference (PLSD) test to determine significanceamong genotypes for each testing session.Auditory startle response. Startle testing was performed using SR-Lab startle chambers (San Diego Instruments) (8). Acousticstimuli was produced by high-frequency speakers controlled bySR-Lab software. A 25-min test session was used, in which pulsevalues were 90, 95, 100, 105, 110, 115, and 120 dB on a 70-dBbackground (B) level. Startle pulses were 40 ms in duration andthe trial types were presented several times in a pseudorandomorder with intervening no stimulus trials to control for baselinemovement. Data are presented as the average startle magnitudemeasured during each acoustic pulse ± the SEM. Statisticalanalysis was performed by two-way ANOVA and Fisher’s PLSDtest to determine significance between genotypes for each testingsession. In the auditory startle test, comparison between geno-types for each dB pulse tested per session was performed byStudent’s t test.Rotarod test. Motor coordination was measured using the accel-erating rotarod test with a Roto-Rod Series 8 (IITCLife Sciences)(9). Mice were placed on the stationary rod, which began toaccelerate from 10 rpm, and the rotations per minute at fall wererecorded. Mice were subjected to six testing trials per day, whichconsisted of two sets of three trials each, with 1 min betweeneach trial and 2 h between each set. Data are presented as av-erage time to fall(s) ± SEM. Statistical analysis was performedby ANOVA and Fisher’s PLSD test to determine significanceamong genotypes.Hanging wire test. Peripheral strength was assessed using thehanging wire test (9). Mice were held so that only their forelimbscontacted an elevated metal bar (12 mm diameter; 37 cm abovethe floor). Latency to fall was measured up to a maximum of30 s; each mouse was tested in three trials separated by a 30-srest period. Data are presented as the average latency to fall(s) ±SEM. Statistical analysis was performed by ANOVA and Fisher’sPLSD test to determine significance among genotypes.Optomotor test.Visual acuity was assessed in the optomotor test asdescribed previously (10) using a stationary elevated platformsurrounded by a drum with black-and-white striped walls. Themouse was placed on the platform to habituate for 1 min and



then the drum was rotated at 2 rpm in one direction for 1 min,stopped for 30 s, and then rotated in the other direction for 1min. The total number of head tracks (15° movements at thespeed of the drum) was recorded.Auditory brainstem response. Brainstem auditory-evoked potentials(BAEPs) were measured in 9- to 10-mo-old ABHD12+/+,ABHD12+/−, and ABHD12−/− mice (n = 5; mixed sex) afterconfirming that ABHD12−/− mice of this age exhibit a reducedauditory startle reflex as described above. Mice were anes-thetized using isoflurane and their heads were placed in a ste-reotaxic apparatus. Head hair was shaved off, and the incisionsite was prepared with ethanol and betadine. An incision wasmade, and the skull was exposed and cleaned. Three stainless-steel screw electrodes were inserted into the skull: two over thehippocampus (2.0 mm posterior and 2.0 mm lateral to bregma)and a third over the cerebellum as a control for signal artifacts.Insulated leads from these electrodes were then soldered toa miniconnector that was cemented to the skull with dentalacrylic. Wounds were sutured closed, topical antibiotic ointmentwas applied, mice were injected s.c. with flunixin, and then themice were recovered in a clean warm cage. Mice were monitoredcontinuously during surgery and until fully recovered and then atleast once per day until the experiment’s end. After surgery, micewere housed in individual cages and allowed a 1–2 wk of re-covery before recordings.To measure the BAEPs, mice were anesthetized using iso-

flurane and then fitted with bilateral polyethylene ear tubesplaced into the external auditory canal. A “Y” connector attachedthe two tubes to the central sound source (Grass InstrumentsAudio Amplifier). A computer program written using NationalInstruments software (“LabView”) for the Macintosh II micro-computer was used to generate the stimuli and to collect thedata. The sound stimulus consisted of a 65- to 80-dB sound-pressure level at a rate of 10 Hz. Raw signals were amplified bya Grass P-511 instrumentation amplifier filtered between 300 and3,000 Hz and averaged using custom software operating on aMacintosh II series computer. Stimuli were delivered binaurally ata frequency of 10 per second for a total of 1,024 stimuli. Onlineaveraging of the signals allowed us to repeat trials within a re-cording session to determine the stability of the evoked responses.Tetrad tests for cannabimimetic activity. Two month old ABHD12−/−

and ABHD12+/+ littermates (n = 5 per genotype, mixed sex)were tested for catalepsy, locomotor activity and core tempera-ture (11). Mice were housed on a 0600 hours/1800 hours light/dark cycle, had ad libitum access to water and food, and werehoused individually overnight before testing. Catalepsy was as-sessed on a 0.7-cm-diameter bar fixed 4.5 cm off of the ground.The mouse was placed with its front paws on the bar, and thelength of time the mouse was immobile on the bar was recorded,if applicable. If the mouse moved off the bar, it was placed backon in the original position. The assay was stopped after 60 s orafter the third time the mouse moved off the bar. NeitherABHD12+/+ nor ABHD12−/− mice displayed any cataleptic be-havior. Locomotor activity was assessed in a clear Plexiglas cage(18 × 10 × 8.5 inches) placed atop a 7 cm × 7 cm grid. Thenumber of grid-lines crossed by the hind limbs in 5 min was re-corded. Rectal temperature was measured by a thermocoupleprobe inserted 1.2 cm into the mouse rectum, and temperaturewas determined using a telethermometer. Antinociceptive re-sponses were assessed in 2- to 3-mo-old male ABHD12−/− andABHD12+/+ littermates (n = 8–9 per genotype) by the thermaltail immersion test at four temperatures (52 °C, 54 °C, 56 °C, and58 °C) over a period of 4 d, with one temperature tested each day.Mice were housed individually for the duration of the testing.Each mouse was handheld and 1 cm of the tail was submergedinto a heated water bath and the latency for the mouse to with-drawal its tail was timed and recorded. Data were compared byStudent’s t test are reported as average values ± SEM.

All procedures were conducted in accordance with theguidelines established by the Department of Agriculture and theNational Institutes of Health in the Guide for the Care and Use ofLaboratory Animals and approved by the Institutional AnimalCare and Use Committee of The Scripps Research Institute.Histological and immunohistochemical analyses. Mice were deeplyanesthetized using isoflurane and perfused with 0.1 M phosphatebuffer (PB), followed by 4% (wt/vol) paraformaldehyde. Perfusedbrains were postfixed in 4% paraformaldehyde overnight, cry-oprotected in 30% (wt/vol) sucrose in 4% paraformaldehyde untilthey sank (∼3 d), and rapidly frozen on dry ice. Coronal 25-μmsections were cut on a Leica CM1850 cryostat. Slide-mountedsections from 18- to 21-mo-old ABHD12+/+ and ABHD12−/−

mice (n = 4) were stained with hematoxylin and eosin (H&E),Luxol Fast Blue (LFB) with a hematoxylin counterstain or cresylviolet (Nissl). Stained sections were imaged using a Evos XLCore light microscope (Advanced Microscopy Group).Immunohistochemistry was performed on frozen, free-floating

cryostat sections (25 μm) as described previously (12). Primaryantibodies were rabbit anti-ionized calcium-binding adaptormolecule (Iba)1 (1/500 dilution; Wako), rabbit anti-human glialfibrillary acid protein (GFAP) (1/500 dilution; Abcam), andrabbit anti-mouse calbindin (1/200 dilution; Abcam). Sectionsfrom 18- to 21-mo-old ABHD12+/+ and ABHD12−/− mice (n = 4)were immunostained for GFAP and calbindin. Sections from 6-,12-, and 18-mo-old mice (n = 3–4 per genotype per age) wereimmunostained for Iba1. Sections were incubated with primaryantibodies in 0.5% (wt/vol) BSA in 0.1 M PB for 48 h at 4 °C,incubated with secondary antibody (anti–rabbit-biotin; VectorLaboratories; 1/300 dilution of 1.5 mg/mL stock) in 0.5% (wt/vol)BSA in 0.1 M PB for 1 h at room temperature and detected withABC Elite Vectastain (Vector Laboratories) for 1 h. Dia-minobenzidine (DAB) (peroxidase substrate kit; Vector Labo-ratories) was used as the chromogen. Sections were washed in0.1 M PB after staining and mounted in VectaMount. Im-munostained sections were imaged using a Leica SCN400 wholeslide scanner.Quantitation of Iba1-positive, enlarged microglia (>200 μm2)

was performed in the cerebellar region depicted by the black boxin Fig. S2G in five matching cerebellar sections per mouse usingImageJ software (National Institutes of Health).

LC-MS Metabolite Profiling. Untargeted metabolomics analysis. Dis-covery metabolite profiling was performed as described previously(13). ABHD12−/− and ABHD12+/+ littermates between 2 and 6mo of age (n = 3–5 per genotype) were anesthetized with iso-flurane and killed by decapitation. Brains were harvested, later-ally sectioned, and immediately submerged in liquid N2. Onefrozen brain hemisphere per mouse was weighed and immedi-ately Dounce-homogenized in 8 mL of 2:1:1 (vol/vol/vol) CHCl3:MeOH:PBS, with 10 nmol of PDA and C12:0 MAGE added asinternal standards for negative- and positive-mode analysis, re-spectively. Homogenates were centrifuged for 10 min at 1,400 ×g. The organic (lower) phase was transferred to a clean vial anddried a stream of N2. The metabolomes were resolubilized in 2:1vol/vol CHCl3:MeOH (120 μL), and 30 μL was injected onto anAgilent 6520 QTOF instrument. LC separation was achievedusing the same solid and mobile phases described above forsubstrate assays. To assist in ion formation, 0.1% (vol/vol) am-monium hydroxide or 0.1% (vol/vol) formic acid was added tothe buffers for negative or positive ionization mode, respectively.The LC method consisted of 0.1 mL/min 0% buffer B for 5 min,a 0.4 mL/min linear gradient over 40 min to 100% buffer B, 0.5mL/min 100% buffer B for 10 min, and 0.4 mL/min equilibrationwith 0% buffer B for 5 min, for an overall run time of 60 min. MSanalysis was performed with an ESI source in scanning modefrom m/z = 50–1,200. The capillary voltage was set to 4.0 kV, andthe fragmentor voltage was set to 100 V. The drying gas tem-



perature was 350 °C, the drying gas flow rate was 11 L/min, andthe nebulizer pressure was 45 psi. Analysis of the LC-MS datawere performed by XCMS (freely available at http://metlin.scripps.edu/xcms) (14), which automatically identifies, matches,aligns, and integrates chromatographic peaks corresponding toendogenous metabolites and identifies m/z values that are sig-nificantly altered in user-defined control vs. experimental data-sets. The XCMS results from two independently performedexperiments were compared and filtered for m/z values that ap-peared in both datasets with >twofold change between genotypes,>30,000 average peak-integration area, LC elution time during thelinear gradient, and P < 0.05. Peak integrations for the m/z valuesthat passed these criteria were checkedmanually and normalized fortissue weight and area of the internal standard. Results are pre-sented as the ratio of the average normalized intensitiesmeasured inthe ABHD12−/− vs. ABHD12+/+ (KO/WT) brain metabolomes. Todetermine the molecular identity of the altered metabolites, theexperimental m/z values were searched against known metabolitemasses in three online databases: Metlin (hosted by the ScrippsCenter forMetabolomics, http://metlin.scripps.edu/metabo_search_alt2.php), the Human Metabolome Database (www.hmdb.ca), andthe Lipid Maps Structure Database (www.lipidmaps.org/data/structure/index.html). Putative assignments were confirmed bycoelution with synthetic standards and/or fragmentation analysis asdescribed below.The relative abundance of known lipid species was determined

by manually extracting the mass corresponding to the (M-H)− or(M+H)+ parent ion (in negative- or positive-ionization mode,respectively), integrating the area under the peak and normal-izing this value for the tissue weight and internal standard in-tensity. Statistical significance was determined by Student’s t test.High mass-accuracy measurements. High mass-accuracy measure-ments of m/z = 552.3, 578.4, 580.4, and 608.4 were achieved byvia Fourier transform ion cyclotron resonance (FT-ICR)-MSusing an Apex II 7.0T FT-ICR mass spectrometer (BrukerDaltonics) coupled to an Agilent 1100 LC. LC was performedas described above for untargeted metabolomics analysis innegative-ionization mode. The FT-IRC-MS system is equippedwith a custom electrospray source with two nebulizers for dual-spray ionization, enabling operation in “lock-mass” mode viathe constant infusion of three compounds [3-β-hydroxy-20-ox-opregn-5-en-17-α-yl sulfate (C21H32O6S), m/z = 411.1847;bradykinin fragment 1–8 (C44H61N11O10), m/z = 902.4530;hexatyrosine (C54H56N6O13), doubly charged ion [M-2H]2−

m/z = 497.1880] for internal calibration. Acquisition time was0.9 s per spectrum. Using these parameters, the 213,000 re-solving power was achieved.Metabolite fragmentation analysis. MS/MS analysis was performedon an Agilent 6520 QTOF instrument with an ESI source, usingthe same LC separation and buffers as described above foruntargeted metabolomic analysis in negative ionization mode.

The m/z values corresponding to the altered endogenous me-tabolites or the parent (M-H)− ions of synthetic standards weretargeted for MS/MS analysis, and MS1 and MS2 spectra wereacquired at a rate of 1.02 spectra per second. The collision en-ergies for LPS was 15, for PS was 25, and for PG was 15. Thecapillary voltage was set to 4.0 kV, and the fragmentor voltage wasset to 100 V. The drying gas temperature was 350 °C, the dryinggas flow rate was 11 L/min, and the nebulizer pressure was 45 psi.Targeted metabolite measurements. Brain MAG levels inABHD12−/−, ABHD12+/−, and ABHD12+/+ littermates (6 moold; n = 5) were measured by multiple reaction monitoring(MRM) methods as described previously (5). Tissue LPS, PS,LPG, PG, LPI, and PI levels in ABHD12−/−, ABHD12+/−, andABHD12−/− littermates (6 mo old; n = 5) were quantified usingsimilar targeted MRM methods. Tissue metabolomes wereprepared as described above for untargeted metabolite analysis,except that 17:1 LPI, LPG, and LPS and 17:0/20:4 PI, PG, andPS synthetic lipids were added as internal standards. A portion(10 μL) of the tissue metabolome was injected onto an AgilentG6410B QQQ instrument. LC separation was achieved using thesolid and mobile phases described above for substrate assays andthe following method: 0.1 mL/min 0% buffer B for 5 min, 0.4mL/min linear gradient from 60 to 100% buffer B over 15 min,0.5 mL/min 100% buffer B for 8 min, and 0.5 mL 0% buffer Bfor 3 min (31 min total run time per sample).Relative brain BMP levels were measured in ABHD12+/+ and

ABHD12−/− mice (2–3 mo old; n = 3) using a method similar toone described previously (15) that allows BMP species to bedistinguished from isobaric PG species. Brain metabolomes wereprepared as described above, and 17:0/20:4 PG was added as aninternal standard. A 10-μL quantity of the solubilized brain lipidswas injected onto an Agilent G6410B QQQ instrument. LC wasperformed using the solid phase described above for substrateassays and an 8-min isocratic gradient of 1 mM ammoniumformate in methanol.MS analysis was performed by targeted MRM with an ESI

source in negative-ion mode for (L)PS, (L)PI, and (L)PG andpositive-ion mode for BMP using the precursor to product iontransitions and collision energies (CEs) listed in Table S6. Thedwell time for each lipid was set to 100ms. The capillary was set to4 kV, the fragmentor was set to 100 V, and the delta electronmultiplier voltage (ΔEMV) was set to –200. The drying gastemperature was 350 °C, the drying gas flow rate was 11 l/min,and the nebulizer pressure was 35 psi. (L)PS, (L)PI and (L)PGspecies were quantified by measuring the area under the peak incomparison with the appropriate unnatural internal standardand normalizing for wet tissue weight. The relative levels of BMPspecies were determined by measuring the area under the peakand normalizing to the area of an internal standard and wettissue weight. Data are presented as the average values ± SEM.The Student’s t test was used to assess significance.

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Fig. S1. Behavioral assessment of ABHD12−/− mice. (A) ABHD12−/− mice (black bars) exhibited modest hypermotility in the open field test at 5–6 mo old, butnot 11–12 or 17–18 mo old, compared with ABHD12+/+ (white bars) and ABHD12+/− (gray bars) littermates (n = 8–9 per group). (B) ABHD12−/− (black triangles)mice at 9–10 mo old displayed a significantly reduced auditory startle reflex compared with ABHD12+/− (gray squares) and ABHD12+/+ (white diamonds) lit-termate controls (n = 5 per group). (C–F) Averaged BAEP waveforms of ABHD12+/+ (black line), ABHD12+/− (teal line), and ABHD12−/− (red line) mice measuredin response to 65 dB (C), 68 dB (D), 70 dB (E), and 80 dB (F) auditory stimuli (n = 5 per group; wave peaks labeled P1 to P7). (G) Visual performance in theoptomotor test was not significantly altered by genetic ABHD12 disruption. Data represent the averages ± SEM. Variance among genotypes was assessed bytwo-way ANOVA and Fisher’s PLSD test. The Student’s t test was used to determine significance for each individual dB pulse tested in B. *P < 0.05; **P < 0.01;***P < 0.001 vs. ABHD12+/+ mice.



Fig. S2. Comparative histological analysis of 18-mo-old ABHD12+/+ and ABHD12−/− mice. No obvious genotypic differences were observed between ABHD12+/+

and ABHD12−/− mice in gross brain organization by H&E staining (A and B), myelin by LFB staining (C and D), neurons by Nissl staining (E and F), astrocytes byGFAP immunostaining (G and H), or Purkinje neurons by calbindin D28k immunostaining (I and J). Representative cerebellar images are shown. Black boxes inA, C, E, G, and I represent the magnified regions in B, D, F, H, and J. (Scale bars: A, C, E, G, and I, 1 mm; B, D, and F, 250 μm; H and J, 100 μm.]



Fig. S3. ABHD12 does not regulate bulk endocannabinoid metabolism and signaling in vivo. (A) Total 2-AG hydrolase activity was equivalent in brainmembrane homogenates from ABHD12+/+ (white bars) and ABHD12−/− (black bars) mice. In contrast, ABHD12−/− brain membrane homogenates showed re-duced 2-AG hydrolysis activity following pretreatment with the selective MAGL inhibitor JZL184 (1 μM) (n = 4 per group). (B) Total brain levels of 2-AG andother nonendocannabinoid MAGs were equivalent in brain tissue from ABHD12+/+ (white bars) and ABHD12−/− (black bars) mice (n = 5 per group). (C–E)ABHD12+/+ (white bars) and ABHD12−/− (black bars) mice exhibited equivalent core body temperature (C), spontaneous activity in an open field (D), andantinociceptive responses to thermal pain (E) (n = 5–9 per group). Data are presented as average values ± SEM. Statistical analysis performed by Student’st test. ***P < 0.001 vs. ABHD12+/+ homogenates treated with JZL184.



Fig. S4. Structural assignment of ABHD12-regulated metabolites. (A–C) MS/MS fragmentation analysis of endogenousm/z = 578.3 (A), 580.4 (B), and 608.4 (C)metabolites confirmed their structures as 22:1, 22:0, and 24:1 LPS species, respectively. (D–G) Comparison of retention time (D and F) and MS/MS fragmentationspectra (E and G) for endogenous metabolites (black traces) and synthetic lipid standards (red traces) confirmed the structures of m/z = 810.5 as 18:0/20:4 PS (Dand E) and m/z = 797.5 as 18:0:20:4 PG (F and G). (H and I) MS/MS fragmentation analysis confirmed the structures of m/z = 808.5 as 18:1/20:4 PS (H) and m/z =775.5 as 18:1/18:0 PG (I).



Fig. S5. ABHD12 hydrolyzes multiple LPS species. (A) ABHD12-transfected HEK293T cell membrane homogenates (gray bars) displayed increased hydrolyticactivity compared with mock-transfected cell membrane homogenates (black bars) toward both 16:0 and 20:4 LPS. (B) ABHD12−/− brain membranes (black bars)displayed significantly reduced hydrolytic activity toward 16:0 and 20:4 LPS compared with ABHD12+/+ brain membranes (white bars). Data are presented asaverage values ± SEM (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001 vs. mock or ABHD12+/+ homogenates (Student’s t test).



Table S1. BAEP amplitudes and latencies of waves I–VII and interpeak latency of waves I–IV for ABHD12+/+, ABHD12+/−, and ABHD12−/−

littermates

Auditory stimuli intensity

65 dB 68 dB 70 dB 80 dB

Average SEM P vs. +/+ Average SEM P vs. +/+ Average SEM P vs. +/+ Average SEM P vs. +/+

Amplitude (υV)Wave I

ABHD12+/+ 0.09 0.01 0.11 0.00 0.09 0.01 0.13 0.02ABHD12+/− 0.09 0.01 NS 0.09 0.01 NS 0.11 0.01 NS 0.15 0.01 NSABHD12−/− 0.09 0.01 NS 0.09 0.01 NS 0.09 0.01 NS 0.13 0.01 NS

Wave IIABHD12+/+ 0.14 0.03 0.20 0.03 0.23 0.05 0.23 0.04ABHD12+/− 0.10 0.01 NS 0.12 0.01 NS 0.17 0.02 NS 0.27 0.03 NSABHD12−/− 0.10 0.01 NS 0.19 0.05 NS 0.12 0.02 NS 0.21 0.05 NS

Wave IIIABHD12+/+ 0.17 0.05 0.20 0.03 0.24 0.03 0.29 0.03ABHD12+/− 0.15 0.02 NS 0.14 0.02 NS 0.19 0.03 NS 0.28 0.03 NSABHD12−/− 0.10 0.01 NS 0.17 0.05 NS 0.18 0.03 NS 0.28 0.07 NS

Wave IVABHD12+/+ 0.21 0.02 0.27 0.04 0.25 0.02 0.25 0.05ABHD12+/− 0.25 0.05 NS 0.23 0.03 NS 0.21 0.05 NS 0.25 0.02 NSABHD12−/− 0.27 0.03 NS 0.21 0.03 NS 0.31 0.03 NS 0.25 0.05 NS

Wave VABHD12+/+ 0.18 0.04 0.25 0.06 0.20 0.07 0.22 0.06ABHD12+/− 0.16 0.03 NS 0.36 0.06 NS 0.19 0.04 NS 0.31 0.04 NSABHD12−/− 0.19 0.02 NS 0.27 0.01 NS 0.26 0.01 NS 0.22 0.05 NS

Wave VIABHD12+/+ 0.22 0.05 0.18 0.02 0.16 0.04 0.31 0.04ABHD12+/− 0.16 0.04 NS 0.20 0.03 NS 0.20 0.04 NS 0.29 0.04 NSABHD12−/− 0.15 0.03 NS 0.20 0.06 NS 0.24 0.05 NS 0.34 0.03 NS

Wave VIIABHD12+/+ 0.14 0.02 0.19 0.04 0.11 0.01 0.21 0.02ABHD12+/− 0.12 0.01 NS 0.11 0.01 NS 0.12 0.00 NS 0.19 0.03 NSABHD12−/− 0.14 0.03 NS 0.12 0.01 NS 0.10 0.01 NS 0.23 0.04 NS

Latency (μs)Wave I

ABHD12+/+ 4.34 0.02 4.36 0.02 4.35 0.02 4.35 0.02ABHD12+/− 4.33 0.02 NS 4.34 0.02 NS 4.33 0.01 NS 4.34 0.01 NSABHD12−/− 4.34 0.02 NS 4.35 0.02 NS 4.34 0.02 NS 4.34 0.01 NS

Wave IIABHD12+/+ 6.09 0.02 6.09 0.02 6.08 0.02 6.09 0.03ABHD12+/− 6.10 0.02 NS 6.10 0.02 NS 6.09 0.03 NS 6.08 0.02 NSABHD12−/− 6.09 0.02 NS 6.08 0.02 NS 6.09 0.03 NS 6.08 0.02 NS

Wave IIIABHD12+/+ 6.96 0.05 6.96 0.05 6.96 0.05 6.97 0.05ABHD12+/− 6.97 0.05 NS 6.97 0.05 NS 6.98 0.04 NS 6.98 0.04 NSABHD12−/− 6.98 0.04 NS 6.97 0.04 NS 6.98 0.04 NS 6.99 0.02 0.0347

Wave IVABHD12+/+ 7.75 0.04 7.75 0.04 7.76 0.05 7.79 0.10ABHD12+/− 7.94 0.05 0.0055 7.76 0.05 NS 7.84 0.02 NS 7.81 0.02 NSABHD12−/− 8.02 0.03 0.0004 8.00 0.03 0.0004 8.01 0.04 0.0007 8.01 0.05 0.0145

Wave VABHD12+/+ 8.99 0.06 8.93 0.03 9.00 0.04 8.99 0.04ABHD12+/− 9.23 0.02 0.002 8.95 0.04 NS 9.09 0.04 NS 9.03 0.02 NSABHD12−/− 9.29 0.04 0.0004 9.03 0.06 NS 9.17 0.03 0.0106 9.18 0.07 NS

Wave VIABHD12+/+ 10.13 0.15 10.13 0.15 10.16 0.05 10.16 0.06ABHD12+/− 10.39 0.07 NS 10.33 0.09 NS 10.20 0.04 NS 10.23 0.06 NSABHD12−/− 10.42 0.11 NS 10.37 0.12 NS 10.36 0.11 NS 10.32 0.08 NS

Wave VIIABHD12+/+ 11.07 0.04 11.09 0.03 11.09 0.04 11.10 0.03ABHD12+/− 11.25 0.02 0.0005 11.13 0.04 NS 11.14 0.04 NS 11.10 0.02 NSABHD12−/− 11.25 0.02 0.0005 11.21 0.02 0.0138 11.20 0.03 0.0469 11.10 0.05 NS



Table S1. Cont.

Auditory stimuli intensity

65 dB 68 dB 70 dB 80 dB

Average SEM P vs. +/+ Average SEM P vs. +/+ Average SEM P vs. +/+ Average SEM P vs. +/+

Wave I-IVABHD12+/+ 3.41 0.05 3.39 0.05 3.41 0.06 3.44 0.09ABHD12+/− 3.61 0.05 0.0096 3.42 0.06 NS 3.51 0.03 NS 3.47 0.02 NSABHD12−/− 3.68 0.04 0.0013 3.65 0.03 0.0022 3.67 0.04 0.0056 3.67 0.05 0.0167

Variance among genotypes was assessed by two-way ANOVA and Fisher’s PLSD test (n = 5 per group).



Table S2. Untargeted lipidomic profiling of ABHD12+/+ and ABHD12−/− brain tissue

Lipid m/z Retention time (min) KO/WT P

MAG16:0 331.3 40.9 0.8 0.317518:1 357.3 41.4 1.0 0.802720:4 379.3 40.1 1.3 0.1006

PC34:0 762.4 52.5 0.9 0.508336:1 788.6 52.2 1.1 0.3244

LPC16:0 496.3 38.8 1.1 0.276718:0 524.4 41.4 1.4 0.096118:1 522.4 39.4 1.2 0.188020:4 544.3 37.5 1.4 0.0775

PE34:1 716.5 44.8 0.9 0.183136:4 738.5 44 1.1 0.434438:5 764.5 44.1 1.0 0.737638:4 766.5 46 1.4 0.0821

LPE16:0 452.3 32.7 0.8 0.122218:1 478.3 33.1 1.0 0.976418:0 480.3 34.9 1.1 0.141820:4 500.3 32 1.0 0.7974

PA32:0 647.5 36 0.8 0.263634:1 673.5 36 1.1 0.727436:4 695.5 35.5 0.9 0.243436:2 699.5 36.7 0.9 0.831238:5 721.5 35.7 1.2 0.536838:4 723.5 36.8 1.3 0.1578

LPA16:0 409.2 24.9 0.8 0.266818:1 435.3 25.1 1.0 0.973418:0 437.3 27.3 1.0 0.902620:4 457.2 24.2 0.9 0.5147

Unassigned718.6 41.2 3.4 0.0002814.6 44.8 5.1 1.8 × e−6

PS34:1 760.5 38.591 0.6 0.009436:2 786.5 38.7 0.6 0.000136:1 788.5 40 0.8 0.115638:5 808.5 38 2.9 0.020938:4 810.5 39.5 3.5 1.7 × e−06

LPS16:0 496.3 29.2 1.5 0.049818:1 522.3 29.5 1.0 0.937018:0 524.3 30.8 3.1 0.000120:4 544.3 28.5 3.5 0.001420:0 552.3 31.3 7.7 0.006822:6 568.3 28.7 1.0 0.842922:1 578.3 31.3 12.0 0.001022:0 580.4 33 38.6 0.000424:0 608.4 34.7 5.9 0.0003

PG32:0 721.5 42.3 0.5 0.001034:1 747.5 42.4 0.9 0.333534:0 749.5 43.2 1.0 0.512736:1 775.5 41.9 3.8 7.5 × e−7

36:4 797.5 41.6 3.0 7.8 × e−6

LPG20:4 531.3 31.3 1.1 0.7069

LPI16:0 571.3 31.8 0.6 0.0368



Table S2. Cont.

Lipid m/z Retention time (min) KO/WT P

18:1 597.3 32 0.9 0.673418:0 599.3 33.7 1.1 0.668720:4 619.3 29.1 2.3 0.012322:6 643.3 31.3 0.9 0.7659

FFA16:0 255.2 30 1.1 0.542318:1 281.2 30.4 1.0 0.948618:0 283.3 31.9 1.1 0.230520:4 303.2 29.5 1.1 0.441122:6 327.2 29.7 1.0 0.832322:0 339.3 35.5 1.2 0.555822:0 339.3 35.5 1.2 0.555824:0 367.4 37.3 1.4 0.418826:0 395.4 39.4 1.3 0.4665

LC-MS profiling of brain tissue from 2- to 6-mo-old ABHD12+/+ and ABHD12−/− mice revealed multiple lipidspecies that were altered in ABHD12−/− brains. Bolded entries were identified as changing by the XCMS algo-rithm (14) and confirmed by manual integration. Other significantly changing metabolites were identified andquantified by manual extraction and integration, respectively. Note that XCMS identified two additional chang-ing metabolites that we could not structurally assign based on the acquired information (m/z = 718.6 and 814.6).MAG, PC, and LPC species were measured in positive ionization mode, and m/z values represent [M+H]+ ions. Allother lipids were measured in negative ionization mode, and m/z values represent [M-H]− ions. Relative me-tabolite abundance in ABHD12−/− vs. ABHD12+/+ brain metabolomes (KO/WT) is presented as a ratio of theaverage integrated peak intensities (n = 3–5 per genotype). Statistical analysis was performed by the Student’st test.MAG,monoacylglycerol; PC,phosphatidylcholine; LPC, lysophosphatidylcholine;PE,phosphatidylethanolamine;LPE, lysophosphatidylethanolamine; PA, phosphatidic acid; LPA, lysophosphatidic acid; PS, phosphatidylserine; LPS,lysophosphatidylserine; PG, phosphatidylglycerol; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; FFA,free fatty acid.

Table S3. Structures and analytical data for representative VLC-LPS lipids elevated in brain tissue from ABHD12−/− mice

Observed m/z

Molecular formula

Theoretical m/z

Mean error (ppm)

Molecular assignment and structure Fold elevation in ABHD12−/−

552.3307 C26H51NO9P− 552.3307 −0.1 20:0 LPS 12.2

578.3471 C28H53NO9P− 578.3463 0.8 22:1 LPS 15.2

580.3619 C28H55NO9P− 580.362 −0.1 22:0 LPS 13.5

608.3941 C30H59NO9P− 608.3933 −1.1 24:0 LPS 15.2



Table S4. Targeted brain lipid measurements

ABHD12+/+ (WT) ABHD12+/− (HET) ABHD12−/− (KO)

Average SEM Average SEM HET/WT P vs. WT Average SEM KO/WT P vs. WT

LPS16:0 0.071 0.006 0.081 0.008 1.133 NS 0.154 0.010 2.165 0.000118:1 1.577 0.058 1.696 0.048 1.075 NS 1.894 0.057 1.201 0.004718:0 2.649 0.204 2.875 0.189 1.085 NS 10.034 1.212 3.788 0.000320:4 0.281 0.038 0.363 0.055 1.294 NS 0.550 0.054 1.958 0.003620:1 0.099 0.013 0.121 0.011 1.222 NS 0.309 0.067 3.113 0.015120:0 0.039 0.002 0.043 0.005 1.100 NS 0.477 0.058 12.210 0.000122:6 4.153 0.563 4.652 0.406 1.120 NS 4.402 1.272 1.060 NS22:4 0.223 0.022 0.253 0.019 1.139 NS 0.552 0.031 2.479 2.4 × e−5

22:1 0.033 0.004 0.037 0.006 1.138 NS 0.496 0.055 15.258 3.2 × e−5

22:0 0.025 0.004 0.030 0.008 1.199 NS 0.334 0.037 13.452 3.7 × e−5

24:1 0.018 0.004 0.021 0.002 1.146 NS 0.461 0.021 25.444 2.6 × e−8

24:0 0.038 0.008 0.082 0.029 2.149 NS 0.584 0.033 15.242 2.3 × e−7

PS16:0/18:1 94.773 10.303 79.906 5.424 0.843 NS 31.328 1.792 0.331 0.000218:1/18:1 505.103 36.916 488.154 19.900 0.966 NS 271.902 5.082 0.538 0.000218:0/18:1 365.492 106.201 375.063 47.663 1.026 NS 314.216 48.558 0.860 NS18:1/20:4 31.150 2.177 39.719 4.028 1.275 NS 52.483 4.192 1.685 0.002018:0/20:4 188.760 36.515 202.917 24.656 1.075 NS 493.122 69.991 2.612 0.009318:1/20:1 8.409 0.220 7.465 1.628 0.888 NS 3.766 0.567 0.448 0.000418:0/20:1 44.418 0.952 44.874 10.071 1.010 NS 20.386 3.254 0.459 0.000518:1/22:6 59.357 3.451 57.741 4.828 0.973 NS 42.689 2.782 0.719 0.016418:0/22:6 1,970.196 97.447 1,810.615 131.369 0.919 NS 1,656.428 135.181 0.841 NS20:1/20:4 0.721 0.079 1.063 0.148 1.476 NS 3.225 0.387 4.475 0.000720:0/20:4 0.854 0.110 0.579 0.123 0.678 NS 2.542 0.341 2.977 0.003020:1/22:6 5.381 0.216 4.913 0.586 0.913 NS 4.608 0.469 0.856 NS20:0/22:6 1.986 0.178 1.513 0.394 0.762 NS 1.530 0.142 0.770 NS22:1/20:4 0.255 0.031 0.225 0.043 0.882 NS 2.315 0.281 9.078 0.000322:0/20:4 0.510 0.071 0.361 0.072 0.708 NS 2.562 0.295 5.022 0.000524:1/20:4 0.149 0.026 0.097 0.018 0.648 NS 1.284 0.143 8.611 0.000324:0/20:4 0.055 0.019 0.069 0.007 1.247 NS 0.299 0.076 5.432 0.0149

LPI16:0 2.476 0.311 1.925 0.218 0.778 NS 1.503 0.178 0.607 0.026518:1 2.885 0.416 3.897 0.391 1.351 NS 2.886 0.390 1.001 NS18:0 10.683 1.749 11.290 0.762 1.057 NS 11.612 2.426 1.087 NS20:4 3.802 0.517 4.844 0.436 1.274 NS 7.144 1.141 1.879 0.0285

PI18:1/20:4 35.863 3.193 38.733 3.370 1.080 NS 32.825 2.977 0.915 NS18:0/20:4 228.008 26.338 266.172 28.842 1.167 NS 315.033 24.808 1.382 0.0428

LPG16:0 0.499 0.036 0.520 0.040 1.044 NS 0.440 0.029 0.882 NS18:1 1.987 0.152 2.230 0.124 1.122 NS 2.027 0.124 1.020 NS18:0 0.269 0.033 0.273 0.047 1.017 NS 0.440 0.027 1.638 0.004020:4 0.571 0.047 0.649 0.051 1.137 NS 0.591 0.074 1.035 NS22:6 0.348 0.033 0.337 0.034 0.968 NS 0.221 0.025 0.636 0.0166

PG16:0/16:0 18.220 2.074 20.034 2.241 1.100 NS 12.211 1.519 0.670 0.047616:0/18:1 60.383 7.054 66.335 6.086 1.099 NS 64.905 8.338 1.075 NS18:0/18:1 4.085 0.287 6.047 0.852 1.480 0.043 19.192 2.566 4.698 0.000418:0/20:4 7.604 0.893 10.831 1.140 1.424 0.045 23.034 3.730 3.029 0.0038

BMP16:0/16:0 10.779 0.669 12.528 2.943 1.162 NS16:0/18:1 12.329 1.129 13.781 3.349 1.118 NS18:1/18:1 3.087 0.037 3.525 0.512 1.142 NS20:4/22:6 2.503 0.349 2.182 0.421 0.872 NS22:6/22:6 25.302 3.456 23.309 3.821 0.921 NS

MRM MS methods were used to quantify (L)PS, (L)PI, and (L)PG levels in ABHD12+/+ (WT), ABHD12+/− (HET), and ABHD12−/−

(KO) brains and measure relative BMP levels in ABHD12+/+ and ABHD12−/− brains. (L)PS, (L)PI, and (L)PG levels are presented asnanomoles per gram of tissue, and relative BMP levels are presented as arbitrary units normalized for tissue weight. Statisticalanalysis was performed by the Student’s t test (n = 3–5 per group). HET, heterozygous; NS, not significant.



Table S5. MRM transition list for targeted metabolite measurements

Class Species Precursor Product CE (V)

PS 17:0/20:4 std 797 269 35PS 16:0/18:1 761 255 35PS 18:1/18:1 787 281 35PS 18:0/18:1 789 283 35PS 18:1/20:4 809 281 35PS 18:0/20:4 811 283 35PS 18:1/20:1 815 281 35PS 18:0/20:1 817 283 35PS 18:1/22:6 833 281 35PS 18:0/22:6 835 283 35PS 20:1/20:4 837 309 35PS 20:0/20:4 839 311 35PS 20:1/22:6 861 309 35PS 20:0/22:6 863 311 35PS 22:1/20:4 865 337 35PS 22:0/20:4 867 339 35PS 24:1/20:4 893 365 35PS 24:0/20:4 895 367 35LPS 17:1 std 508 421 15LPS 16:0 496 409 15LPS 18:1 522 435 15LPS 18:0 524 437 15LPS 20:4 544 457 15LPS 20:1 550 463 15LPS 20:0 552 465 15LPS 22:4 572 485 15LPS 22:6 568 481 15LPS 22:1 578 491 15LPS 22:0 580 493 15LPS 24:1 606 519 15LPS 24:0 608 521 15PG 17:0/20:4 std 784 303 40PG 16:0/16:0 722 255 40PG 16:0/18:1 748 281 40PG 18:0/18:1 776 281 40PG 18:0/20:4 798 303 40LPG 17:1 std 496 267 20LPG 16:0 483 255 20LPG 18:1 509 281 20LPG 18:0 511 283 20LPG 20:4 531 303 20LPG 22:6 555 327 20PI 17:0/20:4 std 872 303 40PI 18:1/20:4 884 303 40PI 18:0/20:4 886 303 40LPI 17:1 std 583 267 40LPI 16:0 571 255 40LPI 18:1 597 281 40LPI 18:0 599 283 40LPI 20:4 619 303 40PG 17:0/20:4 std 803 614 15BMP 16:0/16:0 741 313 20BMP 16:0/18:1 767 339 20BMP 18:1/18:1 793 339 20BMP 20:4/22:6 861 361 20BMP 20:4/22:6 885 385 20

Listed are the precursor and product ion m/z values and collision energies (CEs) used to quantify lipidmetabolite levels in ABHD12+/+, ABHD12+/−, and ABHD12−/− tissues. std, standard.



Table S6. Targeted lipid measurements in dissected brain regions and peripheral tissues



CerebellumLPS

16:0 0.111 0.006 0.152 0.011 1.374 0.0189 0.196 0.012 1.766 0.000818:1 2.351 0.087 2.890 0.120 1.230 NS 2.730 0.273 1.161 NS18:0 11.246 0.677 9.083 1.180 0.808 NS 26.948 0.644 2.396 2.8 × e−6

20:4 0.177 0.016 0.197 0.017 1.116 NS 0.438 0.019 2.471 4.3 × e−5

20:0 0.064 0.010 0.085 0.003 1.330 NS 0.691 0.038 10.838 3.5 × e−6

22:6 2.626 0.419 2.757 0.587 1.050 NS 2.669 0.313 1.016 NS22:1 0.065 0.005 0.046 0.004 0.704 NS 1.091 0.054 16.734 1.4 × e−6

22:0 0.131 0.011 0.116 0.014 0.888 NS 2.089 0.107 16.003 1.8 × e−6

24:1 0.061 0.011 0.049 0.007 0.811 NS 1.500 0.076 24.650 1.4 × e−6

24:0 0.137 0.019 0.111 0.006 0.813 NS 3.318 0.105 24.285 9.4 × e−8

PS16:0/18:1 61.244 3.915 74.716 13.522 1.220 NS 23.781 0.282 0.388 0.000116:0/18:0 71.590 13.549 64.999 15.304 0.908 NS 37.590 2.279 0.525 0.048218:1/18:1 517.582 10.009 607.106 78.046 1.173 NS 274.830 22.587 0.531 0.000118:0/18:1 336.359 75.806 377.764 55.387 1.123 NS 204.420 21.464 0.608 NS18:1/20:4 28.916 1.262 43.833 5.902 1.516 0.0484 55.100 2.130 1.906 4.2 × e−5

18:0/20:4 88.293 7.101 124.897 17.039 1.415 NS 188.531 13.129 2.135 0.000518:1/22:6 43.105 1.575 51.536 6.458 1.196 NS 25.509 1.088 0.592 0.000118:0/22:6 897.481 76.118 1,123.395 160.488 1.252 NS 561.455 51.255 0.626 0.010620:1/20:4 0.620 0.118 0.891 0.120 1.437 NS 2.002 0.147 3.229 0.0003

CortexLPS

16:0 0.146 0.025 0.196 0.033 1.343 NS 0.219 0.016 1.500 0.049818:1 1.102 0.120 1.318 0.172 1.195 NS 1.233 0.049 1.119 NS18:0 12.166 3.685 14.396 5.394 1.183 NS 14.019 1.947 1.152 NS20:4 0.120 0.008 0.160 0.016 1.332 NS 0.344 0.010 2.863 2.4 × e−6

20:0 0.024 0.001 0.023 0.002 0.987 NS 0.177 0.013 7.460 2.6 × e−5

22:6 2.969 0.333 2.530 0.259 0.852 NS 2.814 0.196 0.948 NS22:1 0.021 0.004 0.017 0.002 0.815 NS 0.214 0.015 10.403 1.7 × e−5

22:0 0.027 0.004 0.040 0.002 1.485 0.0176 0.369 0.054 13.668 0.000724:1 0.010 0.000 0.017 0.001 1.778 0.0002 0.221 0.028 22.697 0.000324:0 0.044 0.002 0.039 0.008 0.900 NS 0.507 0.070 11.596 0.0006

PS16:0/18:1 46.771 1.053 46.129 1.190 0.986 NS 21.486 1.221 0.459 4.2 × e−6

16:0/18:0 68.203 5.768 58.983 5.509 0.865 NS 41.079 5.928 0.602 0.016818:1/18:1 158.761 10.077 138.931 8.376 0.875 NS 86.792 6.314 0.547 0.000918:0/18:1 102.713 6.766 111.167 11.961 1.082 NS 61.146 5.068 0.595 0.002718:1/20:4 11.169 0.408 14.228 0.697 1.274 0.0091 18.559 0.994 1.662 0.000518:0/20:4 69.664 1.934 85.710 5.362 1.230 0.0305 138.129 9.346 1.983 0.000418:1/22:6 41.326 1.822 38.363 1.342 0.928 NS 26.065 1.418 0.631 0.000618:0/22:6 1,199.876 42.251 1,120.403 58.824 0.934 NS 772.844 50.032 0.644 0.000620:1/20:4 0.261 0.005 0.318 0.031 1.218 NS 0.855 0.055 3.279 3.8 × e−5

HippocampusLPS

16:0 0.470 0.060 0.349 0.044 0.743 NS 0.358 0.052 0.762 NS18:1 3.378 0.961 2.203 0.193 0.652 NS 2.749 0.259 0.814 NS18:0 37.024 4.127 21.639 7.115 0.584 NS 34.106 6.171 0.921 NS20:4 0.567 0.151 0.504 0.054 0.888 NS 1.051 0.165 1.854 NS20:0 0.080 0.009 0.052 0.004 0.652 NS 0.257 0.064 3.209 0.033022:6 8.515 2.012 6.006 0.513 0.705 NS 6.287 0.935 0.738 NS22:1 0.090 0.019 0.043 0.009 0.478 NS 0.336 0.090 3.747 0.037022:0 0.037 0.003 0.086 0.003 2.348 1.9 × e−5 0.272 0.083 7.443 0.030024:1 0.015 0.001 0.043 0.004 2.985 0.0003 0.205 0.061 14.090 0.020624:0 0.052 0.002 0.083 0.007 1.579 0.0055 0.514 0.136 9.816 0.0145

PS16:0/18:1 73.825 1.721 85.593 6.777 1.159 NS 33.670 1.409 0.456 1.9 × e−6

16:0/18:0 99.131 1.419 89.708 10.206 0.905 NS 82.133 10.680 0.829 NS18:1/18:1 182.607 7.921 192.406 4.236 1.054 NS 128.316 17.495 0.703 0.030118:0/18:1 183.719 17.697 247.958 26.508 1.350 NS 200.222 47.229 1.090 NS



Table S6. Cont.



18:1/20:4 19.185 0.540 27.689 1.266 1.443 0.0008 35.947 2.996 1.874 0.001518:0/20:4 127.128 5.757 182.942 5.424 1.439 0.0004 331.087 39.071 2.604 0.002118:1/22:6 49.644 0.622 58.645 3.654 1.181 NS 35.898 1.769 0.723 0.000318:0/22:6 1,586.937 48.575 1,741.337 48.002 1.097 NS 1,373.584 129.562 0.866 NS20:1/20:4 0.422 0.030 0.636 0.038 1.507 0.0045 2.319 0.470 5.496 0.0069

HeartLPS

18:1 1.843 0.068 1.651 0.170 0.896 NS 1.924 0.252 1.044 NS18:0 77.768 7.491 71.598 4.899 0.921 NS 65.145 5.913 0.838 NS20:4 0.506 0.036 0.619 0.107 1.225 NS 0.807 0.150 1.596 NS20:0 0.875 0.115 0.712 0.048 0.813 NS 0.664 0.047 0.758 NS22:6 0.966 0.160 0.846 0.355 0.876 NS 1.674 0.230 1.732 0.03622:1 0.142 0.009 0.131 0.010 0.922 NS 0.132 0.014 0.930 NS22:0 0.121 0.017 0.099 0.009 0.821 NS 0.107 0.011 0.889 NS24:1 0.033 0.005 0.031 0.003 0.934 NS 0.031 0.003 0.924 NS24:0 0.057 0.011 0.040 0.004 0.699 NS 0.061 0.011 1.073 NS

PS16:0/18:1 1.426 0.231 0.938 0.095 0.658 NS 1.173 0.267 0.822 NS18:1/18:1 3.334 0.116 2.388 0.145 0.716 NS 2.153 0.533 0.646 NS18:0/18:1 50.190 4.172 35.505 4.089 0.707 NS 37.654 9.228 0.750 NS18:1/20:4 5.453 0.732 3.939 0.579 0.722 NS 5.839 1.182 1.071 NS18:0/20:4 77.589 4.351 70.076 5.054 0.903 NS 93.848 17.665 1.210 NS18:1/22:6 7.199 0.460 6.443 0.564 0.895 NS 5.773 0.970 0.802 NS18:0/22:6 260.491 27.944 293.383 25.615 1.126 NS 242.349 57.429 0.930 NS20:1/20:4 0.965 0.056 0.844 0.073 0.875 NS 1.068 0.144 1.106 NS

KidneyLPS

16:0 1.965 0.377 1.340 0.097 0.682 NS 1.961 0.765 0.998 NS18:1 1.406 0.371 1.157 0.069 0.823 NS 0.998 0.228 0.710 NS18:0 15.251 1.737 12.729 0.707 0.835 NS 16.781 4.831 1.100 NS20:4 5.362 1.221 4.399 0.401 0.820 NS 4.095 0.398 0.764 NS20:0 0.085 0.012 0.071 0.006 0.836 NS 0.090 0.014 1.059 NS22:6 1.250 0.208 0.854 0.052 0.683 NS 0.818 0.091 0.654 NS22:1 0.025 0.002 0.026 0.006 1.043 NS 0.038 0.009 1.493 NS22:0 0.051 0.006 0.059 0.005 1.154 NS 0.090 0.012 1.746 0.02424:1 0.019 0.003 0.018 0.002 0.946 NS 0.027 0.004 1.405 NS24:0 0.057 0.006 0.058 0.008 1.005 NS 0.097 0.011 1.697 0.012

PS16:0/18:1 3.958 0.331 4.133 0.282 1.044 NS 2.980 0.329 0.753 NS18:1/18:1 1.517 0.094 1.676 0.119 1.105 NS 1.192 0.118 0.786 NS18:0/18:1 14.476 2.034 16.704 2.029 1.154 NS 12.412 2.033 0.857 NS18:1/20:4 2.749 0.194 3.048 0.123 1.109 NS 2.434 0.231 0.885 NS18:0/20:4 118.126 14.986 135.112 13.935 1.144 NS 111.654 8.472 0.945 NS18:1/22:6 1.173 0.096 1.055 0.047 0.900 NS 0.805 0.121 0.686 0.03518:0/22:6 21.487 2.435 18.406 1.478 0.857 NS 14.373 1.855 0.669 0.04220:1/20:4 0.181 0.021 0.222 0.020 1.229 NS 0.223 0.020 1.236 NS

LiverLPS

16:0 3.840 0.886 2.624 0.340 0.683 NS 4.064 0.816 1.058 NS18:1 1.169 0.231 0.696 0.032 0.595 NS 0.863 0.130 0.739 NS18:0 18.958 2.622 18.402 2.639 0.971 NS 24.338 3.568 1.284 NS20:4 2.939 0.333 2.863 0.261 0.974 NS 4.194 0.482 1.427 NS20:0 0.150 0.041 0.085 0.024 0.565 NS 0.206 0.036 1.371 NS22:6 2.003 0.203 1.596 0.206 0.796 NS 2.117 0.336 1.057 NS22:1 0.030 0.003 0.034 0.010 1.130 NS 0.048 0.010 1.572 NS22:0 ND ND ND24:1 ND ND ND24:0 ND ND ND

PS16:0/18:1 0.907 0.113 0.918 0.060 1.012 NS 0.751 0.023 0.827 NS18:1/18:1 1.315 0.172 1.283 0.106 0.976 NS 1.023 0.039 0.778 NS



Table S6. Cont.



18:0/18:1 5.904 0.716 6.172 1.030 1.045 NS 6.184 0.438 1.047 NS18:1/20:4 8.001 1.149 8.339 1.372 1.042 NS 7.083 0.519 0.885 NS18:0/20:4 157.452 17.089 171.544 14.002 1.090 NS 219.257 10.849 1.393 0.01618:1/22:6 4.448 0.649 3.795 0.771 0.853 NS 3.190 0.251 0.717 NS18:0/22:6 74.678 4.668 70.435 9.342 0.943 NS 73.659 6.121 0.986 NS20:1/20:4 0.508 0.089 0.558 0.184 1.100 NS 0.930 0.146 1.832 0.038

LungLPS

16:0 3.326 0.516 2.700 0.275 0.812 NS 3.609 1.504 1.085 NS18:1 7.763 1.366 6.470 0.303 0.833 NS 7.473 2.517 0.963 NS18:0 81.408 12.081 67.877 8.304 0.834 NS 77.148 32.753 0.948 NS20:4 2.840 0.372 3.117 0.382 1.098 NS 2.963 0.455 1.043 NS20:0 0.618 0.104 0.503 0.061 0.814 NS 0.700 0.272 1.134 NS22:6 1.242 0.200 1.093 0.168 0.880 NS 1.036 0.176 0.834 NS22:1 1.002 0.193 0.891 0.069 0.889 NS 0.740 0.171 0.738 NS22:0 0.549 0.071 0.467 0.036 0.850 NS 0.500 0.081 0.910 NS24:1 0.325 0.059 0.301 0.025 0.928 NS 0.287 0.054 0.883 NS24:0 0.509 0.043 0.525 0.046 1.033 NS 0.629 0.079 1.237 NS

PS16:0/18:1 14.454 2.480 10.744 0.872 0.743 NS 10.380 3.253 0.718 NS18:1/18:1 14.468 1.457 12.083 0.742 0.835 NS 10.888 3.216 0.753 NS18:0/18:1 130.766 23.361 116.473 12.442 0.891 NS 112.326 36.066 0.859 NS18:1/20:4 6.430 0.372 7.590 0.424 1.180 NS 7.240 0.654 1.126 NS18:0/20:4 143.571 22.610 146.537 11.278 1.021 NS 143.625 26.044 1.000 NS18:1/22:6 3.205 0.470 3.861 0.481 1.205 NS 2.800 0.520 0.874 NS18:0/22:6 61.919 7.327 60.503 2.458 0.977 NS 37.238 4.754 0.601 0.02220:1/20:4 0.957 0.152 1.029 0.081 1.076 NS 1.133 0.203 1.185 NS

EyeLPS

16:0 0.655 0.281 0.550 0.166 0.840 NS 0.438 0.152 0.669 NS18:1 3.034 0.373 3.797 0.832 1.251 NS 2.508 0.757 0.827 NS18:0 48.901 7.355 42.534 11.010 0.870 NS 35.497 5.701 0.726 NS20:4 1.175 0.371 1.534 0.466 1.306 NS 1.248 0.438 1.062 NS20:0 0.188 0.045 0.159 0.043 0.848 NS 0.172 0.050 0.918 NS22:6 8.545 2.106 9.648 4.958 1.129 NS 6.302 1.026 0.738 NS22:1 0.179 0.044 0.152 0.030 0.850 NS 0.136 0.048 0.761 NS22:0 0.133 0.030 0.112 0.020 0.845 NS 0.122 0.038 0.919 NS24:1 0.122 0.015 0.095 0.013 0.782 NS 0.104 0.026 0.855 NS24:0 ND ND ND

PS16:0/18:1 22.186 0.335 19.174 1.223 0.864 NS 14.136 0.493 0.637 1.5 × e−5

16:0/18:0 216.782 29.567 187.488 27.274 0.865 NS 253.100 17.644 1.168 NS18:1/18:1 105.734 3.641 100.532 1.602 0.951 NS 97.691 3.926 0.924 NS18:0/18:1 108.142 4.179 102.759 2.817 0.950 NS 99.437 6.479 0.920 NS18:1/20:4 9.985 0.923 10.154 1.034 1.017 NS 10.499 1.239 1.051 NS18:0/20:4 111.826 4.729 128.021 2.501 1.145 NS 137.912 9.969 1.233 NS18:1/22:6 14.354 0.555 13.457 0.243 0.938 NS 13.195 0.744 0.919 NS18:0/22:6 257.629 14.726 258.003 3.144 1.001 NS 232.762 25.471 0.903 NS20:1/20:4 0.482 0.044 0.530 0.042 1.100 NS 0.636 0.060 1.319 NS

MRM MS methods were used to quantify LPS and PS levels in several brain regions (cerebellum, cortex, and hippocampus) andperipheral tissues (heart, kidney, liver, lung, and eye) from ABHD12+/+ (WT), ABHD12+/− (HET), and ABHD12−/− (KO) mice. Lipid levelsare presented as nanomoles per gram of tissue. Statistical analysis was performed by the Student’s t test (n = 5 per group). HET,heterozygous; ND, not detected; NS, not significant.



Table S7. Recombinant ABHD12 substrate assay conditions

Lipid substrate Substrate, μM Product Internal standard

18:1 MAG 100 18:1 FFA (OA) 15:0 FFA (PDA)18:1 LPA 100 18:1 FFA (OA) 15:0 FFA (PDA)18:1 LPC 100 18:1 FFA (OA) 15:0 FFA (PDA)18:1 LPE 100 18:1 FFA (OA) 15:0 FFA (PDA)18:1 LPG 100 18:1 FFA (OA) 15:0 FFA (PDA)18:1 LPI 100 18:1 FFA (OA) 15:0 FFA (PDA)18:1 LPS 100 18:1 FFA (OA) 15:0 FFA (PDA)18:1/18:1 BMP 100 18:1 LPG 17:1 LPG18:1/18:1 DOG 100 18:1 MAG 15:0 MAG16:0/18:1 PA 100 18:1 PA 17:0 LPA16:0/18:1 PC 100 16:0 PC 15:0 LPC16:0/18:1 PE 100 18:1 PE 17:0 LPE16:0/18:1 PG 100 18:1 PG 17:1 LPG16:0/18:1 PI 50 18:1 PI 17:1 LPI16:0/18:1 PS 100 18:1 PS 17:1 LPS

Listed are the substrate concentration, product detected, and internalstandard for each lipid substrate tested. MAG, monoacylglycerol; FFA, freefatty acid; OA, oleic acid; PDA, pentadecanoic acid; LPA, lysophosphatidicacid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPI,lysophosphatidylinositol; LPS, lysophosphatidylserine; BMP, bismonoacylgly-cerolphosphate; DOG, dioleoylglycerol; PA, phosphatidic acid; PC, phospha-tidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI,phosphatidylinositol; PS, phosphatidylserine.



Supporting Information - PNAS dodecylmonoalkylglycerol ether (C12:0 MAGE) were pur-chased from Sigma-Aldrich. Monopentadecanoin (C15:0 MAG) andmonoheptadecanoin(C17:0MAG)werepurchasedfromNu-

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