Accela lc

Ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) was performed on the mitochondrial lipid extracts (10 µL injection volume) using an Accela LC coupled with an Exactive mass spectrometer (ThermoFisher Scientific, Waltham, USA) in positive and negative electrospray ionisation modes (ESI).

An LC-MS system—Accela LC and Exactive Orbitrap MS (Thermo Fisher Scientific, Hemel Hempstead, UK)—was used for metabolomic analysis of degenerated and parental control strains of CM2.

Individual non-polar extracts were dried in a rotary evaporator and reconstituted in 100 μl of isopropanol prior to analysis. Accurate mass LC-MS was performed on lipid extracts (10 μl injection volume) of individual wasps using an Accela LC coupled with an Exactive mass spectrometer (ThermoFisher Scientific, USA) in positive and negative electrospray ionisation modes (ESI). An Ace Excel 2 Super C18 (2 μm particle size, 2.1 × 50 mm) column equipped with an appropriate guard column was maintained at 40 °C with a variable flow rate throughout analysis. The LC gradient program (total duration 10 min) had an initial starting proportion of 62.5% B with a flow rate of 300 μl followed by a linear increase to 99% B with a flow rate of 400 μl over 3 min. This proportion was maintained for a further 5 min followed by a linear decrease to the starting proportion of 62.5% B and flow rate of 300 μl over 2 min. Ions were monitored within the range of m/z 100 to 1500 (ESI voltage: 3500, capillary temperature: 350 °C, scan rate: 250 ms, FT resolution: 25,000). A pooled quality control sample comprising 10 μL from each experimental sample was generated and injected throughout the run. Stability sample replicates were assessed for clustering and drift over time. A set of key ions were selected within each set of replicates and the RSDs of their peak area and retention times.

Accurate mass LC-MS was performed on the mitochondrial lipid extracts (10 μL injection volume) using an Accela LC coupled with an Exactive mass spectrometer (ThermoFisher Scientific, Waltham, USA) in positive and negative electrospray ionisation modes (ESI). An ACE EXL Excel 2μm SuperC18 2.1 x 50mm column equipped with an appropriate guard column was maintained at 40°C with a variable flow rate throughout analysis. The LC gradient program used a water 60%/Acetonitrile 40% (A)-to-water (10%)/acetonitrile (100%)/isopropanol (80%) gradient (B) modified with 0.1% ammonium acetate (30%-100 %B over 12 minutes). Ions were monitored within the range of m/z 100 to 1500 (ESI voltage: 3500, capillary temperature: 350°C, scan rate: 250 ms, FT resolution: 25,000). A pooled quality control sample comprising 5 μL from each experimental sample was generated and injected throughout the run. LC-MS data were aligned and exported using the propriety software Progenesis QI (Nonlinear Dynamics, UK).

High-resolution MS/MS analyses were performed on a C18 Prevail column (150 × 2.1 mm, 2.7 µm) (Grace, Deerfield, IL, USA) eluted with a multilinear gradient made with A (water containing 1% acetonitrile and 2% formic acid) and B (acetonitrile containing 2% formic acid). Gradient elution was as follows: from 97% A to 91% in 5 min, 91% to 85% in 25 min, from 85% to 64% in 35 min, from 64% to 10% in 10 min, and isocratic for 20 min at 200 µL/min flow rate. Five microliters of the sample were injected onto the column kept at 20 °C. The Exactive system was composed of Accela LC coupled to the Orbitrap mass spectrometer and controlled with the Xcalibur software version 2.0.7 (Thermo Fisher Scientific, Austin, TX, USA).