140424_PMA_SC_AP_Wi_DCM_ddMS2_pos:
Chromatographic separation was performed on a Thermo Dionex Ultimate 3000 UHPLC system interfaced to a Q-Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany), using a heated electrospray ionization (HESI-II) source. The LC conditions were as follows: column, Waters BEH C18 150 × 2.1 mm i.d., 1.7 μm; mobile phase, (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid; flow rate, 460 μL/min; injection volume, 3 μL; gradient, linear gradient of 25%−100% B over 30 min followed by an isocratic step of 100% B for 10 min. In positive ion mode, diisooctyl phthalate C24H38O4 [M + H]+ ion (m/z 391.28429) was used as internal lock mass. The optimized HESI-II parameters were as follows: source voltage, 4.0 kV (pos); sheath gas flow rate (N2), 50 units; auxiliary gas flow rate, 12 units; spare gas flow rate, 2.5; capillary temperature, 266.25 °C (pos), S-Lens RF Level, 50. The mass analyzer was calibrated using a mixture of caffeine, methionine-arginine-phenylalanine-alanine-acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1621 in an acetonitrile/methanol/water solution containing 1% acid by direct injection. The data-dependent MS/MS events were performed on the 5 most intense ions detected in full scan MS (Top5 experiment). The MS/MS isolation window width was 1 m/z, and the stepped normalized collision energy (NCE) was set to 20−35−50 units. In data-dependent MS/MS experiments, full scans were acquired at a resolution of 35000 fwhm (at m/z 200) and MS/MS scans at 17500 fwhm both with a maximum injection time of 50 ms. After being acquired in MS/MS scan, parent ions were placed in a dynamic exclusion list for 3.0 s.
050418_LP__P03_35_pos and 050418_LP__P03_36_pos:
Chromatographic separation was performed on a Waters Acquity UPLC system interfaced to a Q-Exactive Focus mass spectrometer (Thermo Scientific, Bremen, Germany), using a heated electrospray ionization (HESI-II) source. Thermo Scientific Xcalibur 3.1 software was used for instrument control. The LC conditions were as follows: column, Waters BEH C18 50 × 2.1 mm, 1.7 μm; mobile phase, (A) water with 0.1% formic acid; (B) acetonitrile with 0.1% formic acid; flow rate, 600 μl·min−1; injection volume, 6 μl; gradient, linear gradient of 5−100% B over 7 min and isocratic at 100% B for 1 min. The optimized HESI-II parameters were as follows: source voltage, 3.5 kV (pos); sheath gas flow rate (N2), 55 units; auxiliary gas flow rate, 15 units; spare gas flow rate, 3.0; capillary temperature, 350.00°C, S-Lens RF Level, 45. The mass analyzer was calibrated using a mixture of caffeine, methionine–arginine–phenylalanine–alanine–acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1621 in an acetonitrile/methanol/water solution containing 1% formic acid by direct injection. The data-dependent MS/MS events were performed on the three most intense ions detected in full scan MS (Top3 experiment). The MS/MS isolation window width was 1 Da, and the stepped normalized collision energy (NCE) was set to 15, 30 and 45 units. In data-dependent MS/MS experiments, full scans were acquired at a resolution of 35,000 FWHM (at m/z 200) and MS/MS scans at 17,500 FWHM both with an automatically determined maximum injection time. After being acquired in a MS/MS scan, parent ions were placed in a dynamic exclusion list for 2.0 s.