P450 17A1 incubation with
17α-hydroxy-[2,2,4,6,6,21,21,21-2H8]progesterone
(1b) in the presence of
18O2 followed by derivatization
and analysis by HRMS.
A, scheme showing the incubation of deuterated
lyase substrate (1b) with P450
17A1 and cytochrome b5 in the
presence of 18O2. The acetic acid product
(3) was derivatized with
the diazoethylpyridine reagent (4)
and analyzed by liquid chromatography-HRMS. B,
ion chromatograms monitoring the various isotopically labeled
acetate products that were derivatized to the pyridylethyl
esters
(5a–h),
with 6 ppm mass tolerance parameter. a,
m/z 171 window
(d3, 18O);
b, m/z 169 window
(d3, 16O);
c, m/z 170 window
(d2, 18O);
d, m/z 168 window
(d2, 16O);
e, m/z 169 window
(d1, 18O);
f, m/z 167 window
(d1, 16O);
g, m/z 168 window
(d0, 18O);
h, m/z 166 window
(d0, 16O).
C, mass spectrum of the
m/z 166.5–171.3 range by selecting
the tR 3.10–3.19-min time
interval in the ion chromatogram corresponding to the pyridine
ester retention time. Shown at m/z 167.0890 is
the peak corresponding to the acetate from background acetic
acid from the natural abundance of 13C isotope
(5i, expected mass,
m/z 167.0896, Δ 3.6 ppm). The peak at
m/z 171.1099 corresponds to the acetate
derived from the enzymatic product
(5a, expected mass,
m/z 171.1093, Δ 4.1 ppm).
D, expansion of the mass spectrum
(m/z 168.95–169.22) (from
C) showing the detection of
d3-labeled acetate with no
18O incorporation
(5b, expected mass,
m/z 169.1051, Δ 5.3 ppm).
E, expansion of the mass spectrum
(m/z 170.95–171.22) from
C showing the presence of
d3-labeled acetate with
18O incorporation
(5a, expected mass,
m/z 171.1093).
p, profile (peaks are shown in profile mode
and not “centroid”). ESI,
electrospray ionization. RT, retention time.
NL, normalized level. More information
about the meaning of the settings can be obtained from the
Xcalibur Qual Browser User Guide (Thermo Scientific).