Targetlynx software

For the untargeted lipidomic approach, a combination of analysis of the variance (ANOVA) and multivariate statistics, including principal component analysis (PCA) and partial, least-squares discriminant analysis (PLS-DA), identified lipids most responsible for differences between fat-1 and WT sample groups. Compounds were identified by database searches as performed by Human Metabolome Database (HMDB) [19] (link) and METLIN [20] (link) as well as by fragmentation patterns, retention times and ion-mobility-derived collision cross sections versus commercially available reference standards, when available. For the targeted approach, mean concentrations and SEM values (n = 5) for each group (fat-1 and WT) were calculated using appropriate internal standards to normalize for variations in sample preparation and MS detection. Univariate analyses (Student's t-tests) were conducted to assess for significance (p-value) and false-discovery rate (FDR) was used to control for multiple comparison. A volcano plot was produced to compare the fold-change versus the p-value for the targeted lipids.
Data processing and analysis was conducted using Progenesis QI Informatics (Nonlinear Dynamics, Newcastle, UK) and MetaboAnalyst 2.0 [21] (link). Data quantification was performed using TargetLynx software (Waters Corporation, Milford, Massachusetts USA).

Fifty-nine individual follicular fluid samples were retrieved during the IVF procedure and stored in glass tubes at −20 °C until assays. BPS glucuronide (BPSG) was quantified—without resorting to a hydrolysis step—using liquid chromatography-mass spectrometry with an Acquity U-HPLC device coupled to a Xevo-TQ triple quadrupole mass spectrometer (Waters, Saint-Quentin-en-Yvelines, France) operated with positive electrospray ionisation and MRM mode. Chromatographic separation was achieved on a Waters Phenyl-Hexyl U-HPLC column (2.1 × 100 mm; 1.6 µm) with an acidified H₂O/AcN gradient elution (0.3 mL/min, 40 °C). Chromatographic data were monitored using Targetlynx® software (Waters Corporation). Briefly, the samples (250 µL) were purified with anionic exchange solid phase extraction (SPE) cartridges using BPS-G d8 (Toronto Research Chemicals) as internal standard. The resulting extract was derivatised with chloride dansyl. All the follicular liquid was quantified within 1 day with a calibration curve that ranged from 0.5 to 50 ng/mL. The accuracy and precision of the assay were evaluated with two series of three quality control (QC) samples at 0.75, 7.5 and 25 ng/mL. The mean accuracy and intra-day CV precision of the assay were 83% and 8%, respectively, and the limit of quantification (LOQ) was set at 0.5 ng/mL.

Metabolite analysis was performed by gas chromatography mass spectrometry (GC–MS). Lyophilized samples (10 mg), the BS, and the DE were extracted with 0.8 mL of acetone:water:acetic acid (80:19:1 v:v:v) containing ribitol at 4 µg/mL, followed by shaking for 10 min at 4°C at 1400 rpm. After centrifugation (20,000 ×g, 5 min), 100 µL of supernatant were collected and dried for 5 h in a SpeedVac vacuum centrifuge.
Analysis and data processing were performed as previously described (Moret et al., 2018 (link)). Samples were derivatized and analyzed using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass spectrometer, as previously described (Fiehn, 2006 (link); Fiehn et al., 2008 (link)). For processing, data files were converted to NetCDF format and analyzed with AMDIS (http://chemdata.nist.gov/mass-spc/amdis/). A homemade retention indices and mass spectra library built from the NIST, Golm (http://gmd.mpimp-golm.mpg.de/), and Fiehn databases and standard compounds was used to identify metabolites. Peak areas were determined with Targetlynx software (Waters) after conversion of the NetCDF file to Masslynx format. AMDIS and Target Lynx in splitless and split 30 mode data were compiled in a single Excel file for comparison. After blank mean subtraction, peak areas were normalized to ribitol and fresh weight.

StromaNKtert cells were treated with vehicle, 20 mM R-2HG or 1 mM Octyl-R-2HG for 48 h and the metabolites were extracted from cells as previously described35 (link). R-2HG was measured using UPLC coupled with a Xevo-TQ mass spectrometer. Liquid chromatography was performed on Acquity UPLC system (Waters) using a BEH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters Corporation). UPLC linear gradient conditions were: 0–3 min, 1% B; 3–5 min, 30% B; 6–8.5 min; 99% B; and 9.5–12 min 99% A [solvent system A: water/formic acid (100:0.1, vol/vol); B: acetonitrile/formic acid (100:0.1, vol/vol)]. The injection volume was 2 μL, and the column temperature was maintained at 35 °C. Mass spectrometry detection was performed by using a Xevo™ Triple Quadrupole MS (Waters Corporation) equipped with an electrospray ionisation (ESI) source operating in negative ionization mode. The online MS analysis was at the Multiple Reaction Monitoring (MRM) mode. Parameters for the cone energy and collision energy for R-2HG are: parent ion 147.02 m/z, daughter ion 129.02 (m/z), cone energy 14 V, collision 12 V. Quantification of R-2HG was done using Target Lynx software (Waters Corporation).