Multiple reaction monitoring analysis by LC-MS

The amounts of GSLs in whole urinary bladder tissues of four mice (two males and two females) and in Epi and SubEpi of human cases 2, 4, and 8 were determined independently via multiple reaction monitoring (MRM) using triple quadrupole LC-MS (Shimadzu LCMS-8060NX) and LabSolutions, version 5.113. Total lipids were extracted from tissue homogenate with 1 ml chloroform/methanol (1:2, by volume) containing 250 nM GlcCer (d18:1/16:0-d3), LacCer (d18:1/17:0), GM3 (d18:1/18:0-d3), and GD3 (d18:1/18:0-d3) as internal standards. After the removal of tissue residue by centrifugation, the supernatant was dried up under nitrogen flow. Total lipids were resuspended with 180 μl of chloroform/methanol (1:2, by volume) and 20 μl 1 N NaOH and incubated at 37°C for 2 h and then dried up under nitrogen flow. GSLs were purified using a reverse-phase column (MonoSpin C18 FF; GL Sciences). The LC conditions are described in “Structural analysis of GSLs by LC-MS” section. The MS conditions were interface nebulizer gas flow rate 2.0 l/min, heating gas flow rate 10 l/min, interface temperature 200°C, dissolved temperature 355°C, DL temperature 250°C, heat block temperature 300°C, drying gas flow rate 10 l/min, probe voltage 1.0 kV, and focus voltage 2.0 kV. MRM transitions and collision energy are shown in Supplemental Table S2 (20). Peak area of each GSLs was integrated using LabSolutions Insight, version 3.8 SP1, and the amount of GSLs is expressed as relative to internal standards indicated in Supplemental Table S2.