Q exactive hybrid quadrupole orbitrap ms

The peptides were first identified by MALDI-TOF/TOF MS (Bruker Daltonics, Billerica, MA), and the fractions were further pooled to average the peptide content. The eluted fractions were transferred to a nano-RP column (5 mm Hypersil C18, 100 mm × 75 mm, Thermo Fisher Scientific, Waltham, MA, USA) mounted in a Prominence nano-HPLC system (Shimadzu, Nakagyo-ku, Kyoto, Japan) and were eluted with ACN gradient from 5% to 40% containing 0.1% formic acid, for 95 min at 400 nL/min. The elutions were directly entered into Q Exactive hybrid quadrupole-Orbitrap MS (Thermo Fisher Scientific), set in a positive ion mode and data-dependent manner with full MS scan from 350 to 6000 m/z, resolution at 70,000 MS/MS scan with higher collision energy dissociation mode, resolution at 17,500.

For HPLC–high-resolution (HR)-ESMS analysis, samples were re-extracted in MeOH (300 µL), transferred to sterile autosampler vials (polypropylene, 250 µL), and held at 20 °C in the autosampler before measurement. HR-ESMS measurements were performed on a Q-Exactive Hybrid Quadrupole-Orbitrap MS after HPLC on a Dionex Ultimate 3000 series instrument (Thermo Fisher Scientific). The HR-MS applies an atmospheric pressure electrospray ionization source (ES) using nitrogen as nebulizer and drying gas. Measurements were performed in positive ionization mode, with a drying gas temperature of 440 °C, spray voltage of 3.5 kV, and a resolution of 70,000 (FWHM). The HPLC system was equipped with a Shodex Asahipak ODP-50 column (4.0 × 125 mm; 5-µm particle size) using mobile phases A [water including 0.1% formic acid (vol/vol)] and B (acetonitrile) under gradient elution conditions: 0–10 min, 10–12.5% B; 10–10.5 min, 12.5–30% B; 10.5–15 min, 30% B; 15–25 min, 30–50% B; 25–30 min, 50% B; 30–30.5 min, 50–10% B; and 30.5–35 min, 10% B. Ions were recorded as protonated [M+H]⁺ form in single-ion monitoring mode and masses set to m/z: 235.1077 (TM) and 334.1550 (TV). Column temperature was 30 °C; flow rate, 0.5 mL⋅min⁻¹; and injection volume, 10 µL.

Frozen liver tissue (approximately 100 mg) was homogenized in glass vials in cold chloroform/methanol (2:1 volume per volume) with internal standards (Avanti Polar Lipids) for each lipid class (Creative Dynamics); the lower phases were collected, and the solvents were evaporated. The samples were reconstituted in isopropyl alcohol/methanol (1:1 volume per volume) for liquid chromatography–mass spectrometry (LC‐MS) analysis on an Ultimate 3000 ultrahigh‐performance LC system coupled to a Q Exactive hybrid quadrupole‐Orbitrap MS (Thermo Fisher Scientific). A Hypersil GOLD C18 analytic column (100 × 2.1 mm, 1.9 μm) was used for separation of lipids. LipidSearch software (Thermo Fisher) was used for lipid molecular species identification and quantification. The lipid classes selected for the search were triacylglycerol, diacylglycerol, ceramide, cardiolipin, and sphingomyelin. Lipid side‐chain composition is denoted by the a:b convention, where a is the number of carbons in the side chain and b the number of double bonds. Cluster 3.0 and Java Treeview programs were used to generate heat maps for visualizing each lipid species. For detailed information, please refer to the Supporting Information.