Lipid standards

Lipid standards were obtained from Avanti Polar Lipids (Alabaster, AL, USA). The solvents used for extraction and for mass spectrometric analyses were of liquid chromatographic grade from Merck (Darmstadt, Germany) and Optima LC-MS grade from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were purchased from Sigma and were of the best available grade. Lipid species were annotated according to the updated comprehensive classification system for lipids [56 (link)].
For lipid extraction we used five pairs of testes per replicate. Five independent replicates were analysed in each genetic background. The samples were sonicated in 400 µl methanol (containing 0.001% butylated hydroxytoluene as antioxidant) for 5 min, shaken for another 5 min, and finally centrifuged at 13 000 r.p.m. for 5 min. The supernatant was transferred into a new Eppendorf tube and stored at −20°C until mass spectrometric analysis.

Metabolite standards were purchased from Sigma-Aldrich (Vienna, Austria) or Carbosynth (Berkshire, UK). Lipid Standards were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), Sigma-Aldrich (Vienna, Austria) or Carbosynth (Berkshire, UK). All lipid and metabolite standards were weighed, dissolved in an appropriate solvent (mostly methanol or water), and a multi-metabolite, as well as a multi-lipid mix, were prepared. The standard reference material (SRM) 1950 metabolites in frozen human plasma was purchased from the National Institute of Standards and Technology (NIST) (Gaithersburg, USA). Acetonitrile (ACN), isopropanol (IPA), methanol (MeOH), and water were of LC–MS grade and ordered from Fisher Scientific (Vienna, Austria) or Sigma-Aldrich (Vienna, Austria). Ammonium bicarbonate, ammonium formate, and ammonium hydroxide were ordered as LC–MS grade eluent additives from Sigma-Aldrich. Formic acid was also of LC–MS grade and obtained from VWR International (Vienna, Austria).

To determine the nLC–ESI–MS/MS run condition, 28 lipid standards from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) were utilized: 12:0-LPC, 16:0-LPC, 18:1-LPC, 12:0/12:0-PC, 13:0/13:0-PC, 16:0/16:0-PC, 18:1/18:0-PC, 20:0/20:0-PC, 14:0-LPE, 18:0-LPE, 12:0/12:0-PE, 14:0/14:0-PE, 18:0/22:6-PE, 12:0-LPG, 15:0/15:0-PG, 18:0-LPS, 18:0/18:0- PS, 16:0/18:2-PI, 18:0-LPA, 12:0/12:0-PA, (18:1)₄-CL, d18:0/12:0-SM, d18:1/24:0-SM, d18:1/12:0-MHC, d18:1/16:0-DHC, d18:1/12:0-Cer, 16:0/18:1-DAG, and 16:0/16:0/18:1-TAG. All solvents used in this study were of HPLC grade. H₂O, CH₃CN, CH₃OH, isopropanol, and methyl-tert-butyl ether were obtained from Avantor Performance Materials (Center Valley, PA, USA), and CHCl₃ was purchased from Sigma-Aldrich (St. Louis, MO, USA). NH₄HCO₂ and NH₄OH (Sigma-Aldrich) were used as ionization modifiers and added to LC mobile phase solutions. All PL standards were diluted with a binary solvent mixture (9:1, v/v, CH₃OH:H₂O). Silica capillary tubes used for capillary columns, and all connections were purchased from Polymicro Technology, LLC (Phoenix, AZ, USA), with all tubes having inner diameters of 20, 50, 75, or 100 μm (outer diameter of 360 μm for all).

FAMEs standard mixture (47885-U) was purchased from Sigma–Aldrich (Copenhagen, Denmark). Lipid standards were obtained from Avanti Polar Lipids (Alabaster, AL). Ammonium acetate and ammonium formate were purchased from Fluka Analytical (Buchs St. Gallen, Switzerland). HPLC grade chloroform and methanol were obtained from Rathburn (Walkerburn, Scotland). n-Hexane was obtained from Fisher Scientific (Slangerup, Denmark).

Lipid standards were from Avanti Polar Lipids (Alabaster, AL, USA). Solvents for extraction and MS analyses were liquid chromatographic grade (Merck, Darmstadt, Germany) and Optima LC–MS grade (Thermo Fisher Scientific, Waltham, MA, USA). All other chemicals were the best available grade purchased from Sigma-Aldrich (Steinheim, Germany).