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.