Analytical Chemistry Chemical Cytometry Quantitates Superoxide

(pH 7.2) containing 1 mM dithiothreitol (DTT) and 0.1 mM
phenylmethyl-sulfonylfluoride (PMSF) was added. Cells were
scrapping off and recovered into an eppendorf tube. Another 500
µL of buffer was added to recover the maximum number of cells.
Cellular lysis was carried out by a successive freeze-thaw
procedure by immersing the tube into liquid nitrogen for 3 min
followed by 3 min in a water bath at 37 °C (all steps repeated
three times). An aliquot of 50 µL was kept aside to quantify
proteins amount by the Bradford protein assay. A 1.45 µL PMSF
aliquot was added to the remaining 1.45 mL sample to reach a
final concentration of 0.2 mM. Then, the sample was ultracentrifuged
(4 °C, 120 000 g, 20 min). The recovered supernatant was
heated at 95 °C for 5 min and ultracentrifuged (4 °C, 120 000 g,
20 min). A 100 µL aliquot of the supernatant was fractionated using
a Superdex peptide HR 10/30 (GE Healthcare, Uppsala, Sweden)
size exclusion column (10 × 300 mm × 5 µm) and isocratic elution
at 0.7 mL · min -1 50 mM TRIS HCl pH 7.4. The SEC column
was cleaned beforehand by flushing with mobile phase containing5mM�-mercaptoethanol
and 2 mM EDTA in order to
remove adsorbed metal ions. The main Cd-containing fraction
(8.75 - 10.73 mL) was collected from nine injections, freezedried,
resolubilized in 900 µL of water and desalted using a 5
mL HiTrap column (GE Healthcare, Uppsala, Sweden) by
elution with NH3aq (pH 8.0) at 1.5 mL · min -1 . The Cd-containing
fraction was heartcut, freeze-dried, and resolubilized in 25 µL
prior to µHPLC analyses. No special precautions, for example,
by Ar or N2 saturation during the sample preparation, were
taken to reduce oxidation of MTs.
RESULTS AND DISCUSSION
ESI MS Analysis of Rabbit Liver MT-2 Standard. As poor
detection limits are the principal drawback of the existing methods
for MT detection, effort has been focus on the optimization of
the signal-to-noise ratio. The choice of the scan mode between
full scan and single ion monitoring (SIM) was first investigated.
In the infusion conditions proper for the ionization of the Cd7-
MT form (5 mM AcNH4, pH6in50%(v/v) MeOH) the S/N
ratio of the most intense peak at m/z 1380.691 (δ 0.3 ppm,
[M+H + ] 5+ ) was 10 times higher in the SIM mode (centered
at m/z 1367.5 ± 85) than in the full scan (m/z 1325-1410)
mode. Similar results were observed for the corresponding apo-
MT signal (m/z 1225.848 (δ 0.3 ppm) in 0.01% FA in 50% (v/v)
MeOH, pH 1.6) for which the S/N ratio was 7 times higher in
the SIM mode (centered at m/z 1230 ± 13) than in the full
scan mode (m/z 1215-1250). Note that in full scan the injection
time was decreased from 3000 to 400 ms in order to limit the
number of concomitant ions entering the ion trap but the MT
peak S/N ratio was not affected.
In order to evaluate the detection limits in the chromatographic
mode taking into account the abundance of the individual
isoforms, the purity of the MT standard available was investigated
by µHPLC-ICP MS. The chromatogram (Figure 2a) obtained in
the conditions of full stoichiometric metalation (seven Cd atoms)
shows two major peaks ( 114 Cd). The contribution of Zn and Cu
is insignificant (