Product characterization was performed
using a Phenomenex Kinetex 2.6 μm C18 100 Å
(50 mm × 2.1 mm) reverse phase column coupled with an Agilent
6540 liquid chromatography/quadrupole time-of-flight mass spectrometer
(LC/QTOF-MS) in positive ion mode. An enzyme reaction mixture comprised
of 300 mM Tris-HCl (pH 8.0), 500 μM acetyl-CoA, 1 mM serotonin,
and 5 μg AANATA in a final volume of 750 μL was incubated
for 30 min at room temperature. Then AANATA was removed from the reaction
mixture by centrifugation using a Millipore 10 kDa filter. The resulting
sample was injected on the LC/QTOF-MS, and the retention time and
high-resolution mass were compared with those of a commercial standard
of N-acetylserotonin. Conditions for LC/QTOF-MS analysis
are described in ref (27 (link)).
using a Phenomenex Kinetex 2.6 μm C18 100 Å
(50 mm × 2.1 mm) reverse phase column coupled with an Agilent
6540 liquid chromatography/quadrupole time-of-flight mass spectrometer
(LC/QTOF-MS) in positive ion mode. An enzyme reaction mixture comprised
of 300 mM Tris-HCl (pH 8.0), 500 μM acetyl-CoA, 1 mM serotonin,
and 5 μg AANATA in a final volume of 750 μL was incubated
for 30 min at room temperature. Then AANATA was removed from the reaction
mixture by centrifugation using a Millipore 10 kDa filter. The resulting
sample was injected on the LC/QTOF-MS, and the retention time and
high-resolution mass were compared with those of a commercial standard
of N-acetylserotonin. Conditions for LC/QTOF-MS analysis
are described in ref (27 (link)).
