Version 2 CD1a protein and HLA-DRB1*03:01 protein (80 μg) were extracted in triplicate in 15 mL glass tubes using chloroform, methanol, and water (72). The organic phase was recovered and dried under nitrogen gas. Eluent residue was redissolved in chloroform-methanol, normalized to 20 μM based on input protein, and stored at –20°C. We injected 20 μL for Q-ToF HPLC-MS positive ion mode analysis using an Agilent 6530 Accurate-Mass Q-TOF mass spectrometer and 1260 series HPLC system with a normal-phase Inertsil Diol column (150 mm × 2 mm, GL Sciences), running at 0.15 mL/minute as described (50, 73, 74).

For the reversed-phase HPLC-MS, an Agilent Poroshell 120 A, EC-C18, 3 × 50 mm, 1.9 μm column coupled with an Agilent EC-C18, 3 × 5 mm, 2.7 μm guard column were used based on published methods (75): mobile phase (A) methanol /water (95/5; v/v) supplemented with 2 mM ammonium-formate and mobile phase (B) 1-propanol/cyclohexane/water (90/10/0.1; v/v/v) supplemented with 3 mM ammonium-formate. The solvent gradient changes in a 20-minute run: 0–4 minutes, 100% A; 4–10 minutes, from 100% A to 100% B; 10–15 minutes, 100% B; 15–16 minutes, from 100% B to 100% A; 16–20 minutes, 100% A. The columns were equilibrated by running 100% A for 5 minutes before the next run. The lipid quantification was determined by the individual ion chromatogram peak area compared with the external standard curves (50).

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