LC-MS/MS Analysis of Sphingolipids and Sterols

Sphingolipids and sterols were quantified using an LCMS-8030 system (Shimadzu). Analytical conditions described for Arabidopsis thaliana by Markham and Jaworski (2007) and Nagano et al. (2014) were optimized for rice sphingolipid species. Chromatographic separation was performed using an XR-ODSII column (Shimadzu; 2.2 μm, 2.0 mm ID, 75 mm) held at 40°C with binary elution gradients consisting of THF/methanol/10 mM ammonium formate (3:2:5) containing 0.1% (v/v) formic acid as solvent A and THF/methanol/10 mM ammonium formate (7:2:1) containing 0.1% (v/v) formic acid as solvent B at a flow rate of 200 μL/min. Each lipid class (Cer, GlcCer, GIPC, SG, and FS) was analyzed separately using solvent concentration gradients, applied linearly over 15 min, as follows: 30 to 100% B for Cer, GlcCer, FS, and SG; 10 to 70% B for GIPC. After elution, the column was washed with 100% solvent B for 1 min and reequilibrated with the appropriate starting solvent for 3 min before the next run.

Lipid species were detected by multiple-reaction monitoring of the transitions of precursor ions [M+H]⁺ to main product ions. Major components of rice sphingolipids containing fatty acids with C20, C22, and C24 carbon length (Watanabe and Imai, 2011) were targeted. To compensate for differences in the MS responses of endogenous sphingolipid species and the internal standards added to samples, sphingolipid classes were fractionated from rice and MS response factors of each internal standard to endogenous species were determined on the basis of their LCB content according to Markham and Jaworski (2007). GIPC content is shown as the total of four subclasses with different sugar moieties (Supplemental Data Set 4). MS response factors for FSs and glucosides were determined using commercially available standards. The m/z values of multiple-reaction monitoring transitions and collision energy were optimized for each compound. Detailed MS/MS parameters for targeted species are listed in Supplemental Data Set 4. The other general conditions were as follows: capillary voltage, 4.5 kV; desolvation gas flow, 10 L/min; nebulizer gas flow, 0.2 L/min; conversion diode voltage, 6 kV; source temperature, 300°C; and collision gas flow, 230 kPa.