Quadrupole orbitrap mass spectrometer

Caudal epididymal spermatozoa (from mice at age of 9–14 weeks) were incubated in mHTF medium for 2 h, collected by adsorption to a polytetrafluoroethylene membrane filter (pore size of 1.0 μm, Merck), and lysed by the addition of ice-cold methanol. The samples were vigorously agitated with a vortex-mixer for 1 min, subjected to ultrasonic treatment for 5 min, and maintained at –30 °C for 1 h. After addition of an internal standard (10-camphorsulfonic acid), the samples were mixed with chloroform, subjected to extraction by ultrasonic treatment for 5 min, and centrifuged at 16,000 × g for 5 min at 4 °C. The upper phase was collected, mixed with chloroform and water, and centrifuged again at 16,000 × g for 5 min at 4 °C. The upper phase was collected and subjected to ultrafiltration at 9100 × g for 60 to 90 min at 4 °C with a UFC3LCCNB-HMT device (Human Metabolome Technologies), the filtrate was dried in an evaporator, and the resulting residue was dissolved in water. Metabolomics analysis of polar metabolites (organic acids and nucleotides) was performed by ion chromatography with a Dionex ICS-5000⁺ HPIC system (Thermo Fisher Scientific) and Dionex IonPac AS11-HC-4 µm column (inner diameter of 2.1 mm, length of 150 mm, particle size of 4 µm; Thermo Fisher Scientific) followed by MS with a quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific)^{52 (link)}.

Non-esterified fatty acids in plasma were analysed using the standard fatty acid kit (Sigma-Aldrich) following manufacture’s instruction. The concentrations of non esterified fatty acids were similar in both studied groups (Table 1).
The total fatty acid content was assessed using gas chromatography after methylating them. The samples (0.1 mL) were mixed with 1.9 mL of chloroform/methanol (2:1 v/v) and 1 mL of cold water, as described before [36 (link)]. Lipids were transferred to sterile glass tubes; chloroform was evaporated under nitrogen. Then, 2 mL of methanolic 5% hydrochloric acid was added. After shaking, the mixture was incubated for two h at 100 °C. After cooling to room temperature, the methyl derivatives were extracted twice with 2 mL of n-hexane, dried under nitrogen, and finally dissolved for analysis. Methylated samples were subjected to a GC/MS system in a quadrupole-orbitrap mass spectrometer (Thermo Scientific). The GC was a Hewlett Packard with a WCOT capillary column, Supelco-10, 35 m × 0.5 mm, 1 μm film. The samples were processed at an initial temperature of 100 °C, and subsequently, processing temperature was increased to 240 °C. The samples, 1 µl, were injected to the GS/MS. Mass spectra identification was set in the selected ion monitoring mode. The standard curve for each fatty acid was performed, and C17:0 served as an internal standard.

Lipid samples were loaded onto a high‐performance liquid chromatograph (HPLC) Ascentis^® Express C18 Column (15 cm, 2.1 mm, 2.7 µm) with a C18 (octadecyl) phase as matrix active group on a fused‐core particle platform and 90 Å pore size. For reversed‐phase chromatography, an HPLC Thermo Scientific Accela 600 LC System instrument was used to elute the lipids onto Q‐Exactive Orbitrap mass spectrometer, which is a hybrid quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific), with a resolving power up to 140 000 m/z 200, and internal mass accuracy of <1 ppm RMS and external <5 ppm RMS. The lipid samples were kept at −80°C until use and then resuspended in chloroform:methanol (1:1, v/v). Solvent A was a mixture of methanol:water:formic acid 0.2% in a 10 mmol L⁻¹ ammonium acetate solution, and solvent B was a mixture of methanol:chloroform:formic acid 0.2% in a 10 mmol L⁻¹ ammonium acetate solution. The lipids were gradient eluted following a 15 minutes 35%‐100% of solvent B gradient at a 260 μL/min flow rate. The mass spectrometer was operated both in positive and negative modes, within a range of collision energies.