LC-MS/MS analysis

For LC-MS/MS analysis, LC-coupled, triple quadrupole mass spectrometers (Xevo TQ-S and Xevo TQ-XS; Waters) were used. For the analyses of seminolipids, SGalDAGs, and sulfatides, the samples were dissolved in chloroform/methanol (1:2, v/v) and separated using a YMC-Triart C18 metal-free reversed-phase column (1.9 μm particle size, 2.1 mm inner diameter, 50 mm length; YMC, Kyoto Japan). The column temperature was set at 55 °C. The flow rate was set to 0.25 ml/min in a binary gradient system using mobile phase A (methanol/acetonitrile/water [1:1:3, v/v] containing 5 mM ammonium formate) and mobile phase B (2-propanol/water [49:1, v/v] containing 5 mM ammonium formate). The gradient steps were as follows: 0 to 1 min, 0% B; 1 to 5 min, linear gradient to 50% B; 5 to 25 min, linear gradient to 95% B; 25 to 25.1 min, step to 0% B; and 25.1 to 30 min, 0% B.

For the analysis of ceramides, sphingomyelins, and DAGs, the samples dissolved in chloroform/methanol (1:2, v/v) were separated using an ACQUITY UPLC CSH C18 reversed-phase column (1.7 μm particle size, 2.1 mm inner diameter, 100 mm length; Waters). The LC flow rate was 0.3 ml/min in a binary gradient system using mobile phase C (acetonitrile/water [3:2, v/v] containing 5 mM ammonium formate) and mobile phase D (acetonitrile/2-propanol [1:9, v/v] containing 5 mM ammonium formate). The gradient steps were as follows: 0 min, 40% D; 0 to 18 min, linear gradient to 100% D; 18 to 23 min, 100% D; 23 to 23.1 min, step to 40% D; and 23.1 to 25 min, 40% D.

Electrospray ionization was performed using the parameters listed in Table S1. MS/MS analysis was performed in multiple reaction monitoring mode using the m/z values of the precursor (Q1) and product (Q3) ions specific to each lipid species and optimized collision energies (Tables S2−S10). Data analysis was performed using the MassLynx software (Waters). The quantity of each seminolipid and SGalDAG species was presented as peak area because their standards were not commercially available. The quantity of each sulfatide, ceramide, sphingomyelin, and DAG species was calculated from its peak area as its ratio to the value of the corresponding C17:0 sulfatide (external standard; Avanti Research), d₉-C16:0 ceramide (internal standard), d₉-C18:1 sphingomyelin (external standard; Avanti Research), and C15:0/d₇-C18:1 DAG (internal standard; Avanti Research), respectively.