Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 3. Comparison of CID and HCD fragmentation modes for the acquisition of sequence information for rabbit liver MT-2a isoform (6.7
µg · mL -1 in 0.01% formic acid, MeOH 50%) directly introduced at 4 µL · min -1 . CID: 40% energy; HCD: 50% energy. Mass range scanned: m/z
335-2000; activation time: 30 ms; injection time: 4000 ms; resolution: 100 000.
smaller than that of 1 order of magnitude observed elsewhere 44
possibly because of the use of a more efficient heated ionization
source.
Mass spectra of the individual MT isoforms [M+H + ] 5+
contributing to the TICs are given in Figure 1-ESI (Supporting
Information). The data are summarized in Table 1. Five MT-2
subisoforms (a-e) could be tentatively identified as N-acetylated
species. Non-acetylated subisoforms MT-2a and MT-2c could also
be detected as minor species. The identification was based on
the accurate mass determination which could be achieved for the
apo-forms with sub-ppm accuracy. The exception was two isoforms
(4 and 7) which were not detected in the ICP MS chromatogram
and for which the measured mass accuracy was 1.5 and 1.2 ppm,
respectively. The achieved mass accuracy allowed the online
determination of the amino acid composition using only few pmole
of MT. The subtraction of the molecular masses of the apo forms
from the corresponding metalated isoforms, the latter determined
with the mass accuracies between 0.1 and 3.7 ppm, allowed the
identification of 20 metal complexes with different rabbit liver
isoforms (Table 1). The overall data confirms the low purity of
the commercial rabbit liver MT-2 preventing its use for most
metalation studies. Note that all the MT-complexes detected could
be efficiently converted to the apo-form by postcolumn acidification
which demonstrates the efficiency of the manifold employed.
(44) Polec, K.; Mounicou, S.; Chassaigne, H.; Lobinski, R. Cell. Mol. Biol. 2000,
46, 221–235.
6952 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
ESI MS Detection Limits. In order to assess the detection
limit of the method an amount of 4.5 ng (ca. 750 fmol, 9-times
less than in Figure 1) of MT-2 standard was analyzed. The five
predominant MT subisoforms could be detected with S/N ratios
comprised between 3 and 66 depending on the MT-isoform
abundance. No significant degradation of the mass accuracy was
observed as metalated isoforms could be measured with mass
accuracies below 3 ppm. Taking the N-Ac-MT2b as example (the
only pure isoform in Figure 1a accounting for ca. 300 fmol of MT)
which was detected with a S/N ratio of 100, the detection limit
(MT concentration producing the signal superior to 3 times
standard deviation of the number of counts on a given mass in
the blank chromatogram) of the apo-isoform could be estimated
as 9 fmol and that of the metalated isoforms for 45 fmol. These
detection limits are by far the lowest ever reported for MT analysis
(500-fold lower than with a former-generation triple quad 44 or with
a recent TOF MS. 28 The HPLC- ICP MS detection limit could be
estimated, for the N-Ac-MT2b subisoform, as 100 fmol of protein
(S/N ) 120 for 4.5 pmol) and was higher than that of ESI MS.
Note that the ESI MS data discussed above were obtained in
the SIM scan mode. When repeated in the full scan mode, the
S/N ratio was found to decrease 2-7 times depending on the
isoform. Mass accuracies obtained for both scan modes were
essentially similar.
Online Sequencing of MT Isoforms. Although the accurate
mass is a valuable parameter for the identification of the MT