Instrumental conditions
Instrumental analysis was carried out using an Ultimate 3000 UHPLC system coupled to an LTQ Orbitrap Velos Pro mass spectrometer system (Thermo Fisher Scientific, San Jose, CA, USA) with a heated electrospray ionization (HESI) source. The same instrumental conditions and methods were used for the plasma and tumor tissue metabolomics and lipidomics analyses.
For the metabolomics analysis, an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters, Milford, MA, USA) was used for the chromatographic separation by maintaining the autosampler and column oven temperature at 4 °C and 50 °C, respectively. Formic acid (0.1%) in distilled water (v/v, mobile phase A) and methanol (v/v, mobile phase B) was used as the mobile phase and eluted at a flow rate of 0.4 mL.min-1throughout the entire analysis. The elution gradient was regulated as follows: the elution started with 100% A and was maintained for 1 min, then gradually decreased to 80% A over next 4 min, and a linear decrease of mobile phase A was made from 80% to 30% from 4 to 10 min. Mobile phase A was then decreased to 0% at 14 min followed by a rapid increase to the initial conditions for re-equilibration at the initial conditions for 2 min.
For the lipidomics analysis, chromatographic separations were performed using an ACE Excel 2 Super C18 column (2.1 × 100 mm, 1.7 µm, Advanced Chromatography Technologies Ltd., Aberdeen, Scotland, UK), and the autosampler and column oven temperature were maintained at 4 °C and 50 °C, respectively. The mobile phase was composed of 10 mM ammonium acetate in either 40% acetonitrile (v/v, mobile phase A) or acetonitrile: isopropanol (10:90, v/v, mobile phase B) and eluted at the same flow rate as that of the metabolomics study. The elution gradient was initiated using 60% A that was maintained for 1min, and from 1 to 3 min, the concentration of A decreased to 35%. Over the next 2 min, mobile phase A decreased to 15%; then, over the next 4 min, it further decreased to 0%, and this condition was maintained for 3 min. Finally, mobile phase A was rapidly increased to reach the initial conditions for 0.5 min (60% A) and re-equilibrated for 3.5 min. The injection volume was 5 µL for all analyses.
Mass spectrometric (MS) conditions were the same for the metabolomics and lipidomics analyses of the plasma and tumor tissue samples, respectively. MS detection was operated in both positive and negative modes using a full scan ranging from m/z 50 to 1600, and data were acquired in centroid mode at a resolution of 60,000. High-energy collision dissociation mode was employed for the dissociation of metabolites using a normalized collision energy of 35% with an isolation width of 1 m/z and an activation time of 10 ms. The detailed MS parameters were as follows: heater temperature, 40 °C; sheath gas flow rate, 45 arb; auxiliary gas flow rate, 10 arb; spray voltage, 4 kV; capillary temperature, 320 °C; and S-lens RF level, 61%. For both the sheath gas and auxiliary gas nitrogen was used.