After dissolving the dried intracellular
methanol extracts in 100 μL HPLC–H2O, 50 μL
were used for SPE and targeted MS analysis of small chain acyl CoA
molecules using a 2-(2-Pyridyl)ethyl silica gel-based SPE column (Supelco,
Merck, Sigma-Aldrich, Germany) and hydrophilic interaction liquid
chromatography (HILIC) coupled to single ion monitoring (SIM) MS analysis.19 (link) For SPE extraction, samples were filled up to
1 mL with equilibration buffer (45% ACN, 20% H2O, 20% Acetic
Acid, 15% Isopropanol (v/v), pH 3). SPE columns were equilibrated
with 1 mL of equilibration buffer (45% ACN, 20% H2O, 20%
Acetic Acid, 15% Isopropanol (v/v), pH 3). After equilibration, samples
were loaded onto the SPE column and washed with 1 mL of the equilibration
buffer. Analytes were eluted from the SPE columns with 2 mL of MeOH/250
mM ammonium formate (4 + 1 v/v, pH 7). The eluates were dried using
a rotary vacuum evaporator (Eppendorf Concentrator Plus, Eppendorf,
Hamburg, Germany). The dried samples were dissolved in 40 μL
of 50% ACN. For HILIC-SIM-MS analysis, 1 μL of sample was injected
on an UHPLC system (Vanquish Flex Quarternary UHPLC System, Thermo
Scientific, Bremen, Germany) equipped with an amide HILIC column (Aquity
UPLC BEH Amide, 130 Å, 1.7 μm, 2.1 × 150 mm, Waters,
Germany). The UPLC was coupled via an electrospray-ionization (ESI)
source to a quadrupole Orbitrap (QExactive HF-X, Thermo Scientific,
Bremen, Germany). HILIC separation was performed using a gradient
from 95 to 50% B in 8 min, and then from 50 to 10% B in 2 min (A:
10 mM NH4Ac in H2O, pH 10; B: 95% ACN, 5% 10
mM NH4Ac in H2O, pH 10). SIM-MS analysis was
carried out in positive mode using a resolution of 60,000 fwhm at
200 m/z, a maximum injection time
of 80 ms and an AGC target of 5 × 104, and the following
SIM isolation windows: acetyl-CoA: m/z 810.1330 ± 15, propionyl-CoA: 824.1487 ± 15, malonyl-CoA:
854.1229 ± 15, succinyl-CoA: 868.1385 ± 15.
Data analysis
was performed in TraceFinder 5.0 (Version 5.0.889.0, Thermo Scientific,
Bremen, Germany). Peaks were fitted using the Genesis algorithm with
the following parameters: percent of highest peak: 1, minimum peak
height (signal/noise): 3, signal-to-noise threshold: 2, tailing factor:
1. Peak integration was manually corrected if necessary. Data were
further processed using R (version 4.0.3) and RStudio (version 1.4.1106)
as described above in the “polar metabolite” section.
methanol extracts in 100 μL HPLC–H2O, 50 μL
were used for SPE and targeted MS analysis of small chain acyl CoA
molecules using a 2-(2-Pyridyl)ethyl silica gel-based SPE column (Supelco,
Merck, Sigma-Aldrich, Germany) and hydrophilic interaction liquid
chromatography (HILIC) coupled to single ion monitoring (SIM) MS analysis.19 (link) For SPE extraction, samples were filled up to
1 mL with equilibration buffer (45% ACN, 20% H2O, 20% Acetic
Acid, 15% Isopropanol (v/v), pH 3). SPE columns were equilibrated
with 1 mL of equilibration buffer (45% ACN, 20% H2O, 20%
Acetic Acid, 15% Isopropanol (v/v), pH 3). After equilibration, samples
were loaded onto the SPE column and washed with 1 mL of the equilibration
buffer. Analytes were eluted from the SPE columns with 2 mL of MeOH/250
mM ammonium formate (4 + 1 v/v, pH 7). The eluates were dried using
a rotary vacuum evaporator (Eppendorf Concentrator Plus, Eppendorf,
Hamburg, Germany). The dried samples were dissolved in 40 μL
of 50% ACN. For HILIC-SIM-MS analysis, 1 μL of sample was injected
on an UHPLC system (Vanquish Flex Quarternary UHPLC System, Thermo
Scientific, Bremen, Germany) equipped with an amide HILIC column (Aquity
UPLC BEH Amide, 130 Å, 1.7 μm, 2.1 × 150 mm, Waters,
Germany). The UPLC was coupled via an electrospray-ionization (ESI)
source to a quadrupole Orbitrap (QExactive HF-X, Thermo Scientific,
Bremen, Germany). HILIC separation was performed using a gradient
from 95 to 50% B in 8 min, and then from 50 to 10% B in 2 min (A:
10 mM NH4Ac in H2O, pH 10; B: 95% ACN, 5% 10
mM NH4Ac in H2O, pH 10). SIM-MS analysis was
carried out in positive mode using a resolution of 60,000 fwhm at
200 m/z, a maximum injection time
of 80 ms and an AGC target of 5 × 104, and the following
SIM isolation windows: acetyl-CoA: m/z 810.1330 ± 15, propionyl-CoA: 824.1487 ± 15, malonyl-CoA:
854.1229 ± 15, succinyl-CoA: 868.1385 ± 15.
Data analysis
was performed in TraceFinder 5.0 (Version 5.0.889.0, Thermo Scientific,
Bremen, Germany). Peaks were fitted using the Genesis algorithm with
the following parameters: percent of highest peak: 1, minimum peak
height (signal/noise): 3, signal-to-noise threshold: 2, tailing factor:
1. Peak integration was manually corrected if necessary. Data were
further processed using R (version 4.0.3) and RStudio (version 1.4.1106)
as described above in the “polar metabolite” section.
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