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