Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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m/z 1172.935 was observed for Ser14 residue (theoretical m/z<br />
1172.939, z ) 4, δ 3.1 ppm) which differentiated this sequence<br />
from the one of MT-1f which contains a Thr14 residue. As the<br />
sequence matched 98% of pig MT-1 sequence and has never been<br />
reported before, it was denoted here as MT-1g. The sequence<br />
coverage with y ions was 88.5%. Consequently, the subisoform<br />
eluting at 37.4 min (5982.149 Da, Figure 6c) was referred to as<br />
N-Ac-MT-1g.<br />
Regarding the identification of the remaining pair of acetylated<br />
and non-acetylated isoforms eluting at 35.0 min (5944.149<br />
Da) and 38.5 min (5986.179 Da), the HCD spectrum of the<br />
N-acetylated form (m/z 1198.243, charge +5) gives the sequence<br />
information. It was found identical to MT1c until Pro38<br />
and then residues Ala39, Arg43, Gln46, Ile49 of MT1c were,<br />
respectively, replaced by Val39, Lys43, Lys46, Val49 as m/z<br />
1098.4547 (theoretical m/z 1098.4550, δ 0.2 ppm), m/z 933.3862<br />
(theoretical m/z 933.3869, δ 0.7 ppm), m/z 781.813 (theoretical<br />
m/z 781.816, δ 3.2 ppm) and m/z 1274.494 (theoretical m/z<br />
1274.499, δ 4.1 ppm) were detected at +2 charge state and the<br />
last one at +1 charge state. An ExPASy database search<br />
indicated that these modifications on 46 and 49 amino acids<br />
residues were reported for MT of others organisms (pig (in<br />
MT3), sheep, bovine and human). The sequence coverage was<br />
92%. As this MT has not been reported before, this pair of<br />
isoforms was denoted as MT-1h and N-Ac-MT-1h, respectively.<br />
The composition of the complexes formed indicates a release<br />
of Cd ions from the nanoparticles and their complexation by<br />
MTs formed.<br />
The identification of the last peak in the µHPLC-ICP MS<br />
chromatogram (peak 8 at 44.3 min (cf. Figure 4b) was less<br />
straightforward as no apo-isoform could be obtained. The presence<br />
of the fragment sequence 7 Cys-Ala-Ala 9 among +4 charge state<br />
fragments is characteristic for an MT-2 class isoforms. Another<br />
part of an MT sequence could be identified among +2 charge<br />
state fragments as 37 Cys-Pro-Val-Gly-Cys-Ala-Lys-Cys-Ala 45 (δ<br />
3.1 ppm), confirming the identity of the MT subisoform as<br />
MT2a. The end of the MT sequence 46 Gln-Gly-Cys-Ile-Cys-Lys-<br />
Gly-Ala-Ser-Asp 55 is observed as +1 charge state (δ 2.3 ppm).<br />
The metallothionein is obviously metalated and the mass<br />
difference in comparison with the N-Ac-MT2a theoretical mass<br />
(6011.190 Da) corresponds to the addition of Cu2Cd2 (351.665<br />
Da) with a loss of 8H + . This state of metalation is corroborated<br />
by the observation of three y ions corresponding to the 7 Cys-<br />
(47) Meloni, G.; Faller, P.; Vasak, M. J. Biol. Chem. 2007, 282, 16068–16078.<br />
(48) Salgado, M. T.; Stillman, M. J. Biochem. Biophys. Res. Commun. 2004, 318,<br />
73–80.<br />
Ala-Ala 9 fragment (m/z 1417.393, m/z 1391.644 and m/z<br />
1373.883 charge +4) differing by 85.89 mass unit from the<br />
corresponding apo subisoforms. The isotopic pattern was<br />
similar to the theoretical isotopic pattern of fragments with<br />
Cu2Cd2 adduct. Therefore peak 8 (Figure 4b) was identified as<br />
N-terminal acetylated Cu2Cd2-MT2a (6355.799 Da, theoretical<br />
6355.792 Da, δ -1.0 ppm) under acidic conditions and as<br />
N-terminal acetylated Cu2Cd6-MT2a (6797.354 Da, theoretical<br />
6797.344 Da, δ -1.4 ppm) under neutral conditions. The late<br />
elution of a similar complex from a rat kidney extract was<br />
reported elsewhere. 23 The comparison of the molecular mass<br />
of the complex with that of the apoMT indicates the presence<br />
of a disulfide bridge formed by oxidation of Cys by Cu(II)<br />
leading to the loss of additional two protons. 17,47 Indeed, the<br />
product ion mass spectra of Cd2Cu2MT2-a (cf. Figure 6), show<br />
a non-fragmented region (from Gly10 until Cys37) of the sequence<br />
suggesting the binding of four metal ions to Cys. This binding<br />
site is shared between the R and � domains of the MT2-a which<br />
is in agreement with the hypothesis of Vaher et al. 17 We can also<br />
notice that the unfragmented central region binding metals<br />
contains 11 Cys like the R domain of MT2a which would explain<br />
the stabilization of Cd2Cu2-MT2a complex. 48<br />
CONCLUSIONS<br />
µHPLC with the combined elemental detection by ICP MS, subppm<br />
molecular mass determination by electrospray orbital trap MS<br />
in conditions favoring metalation and demetalation, and HCD<br />
fragmentation allowing online protein sequencing was demonstrated<br />
as the, to date, most comprehensive approach to the characterization<br />
of metallothioneins in a biological cytosol. The detection limits down<br />
to the low femtomole levels allowed the characterization of metallothionein<br />
subisoforms induced in cell cultures exposed to CdS<br />
nanoparticles confirming the presence of known and the identification<br />
of new MT subisoforms.<br />
ACKNOWLEDGMENT<br />
The contribution of the Region of Aquitaine and the FEDER<br />
funds via CPER A2E (31486/08011464) project is acknowledged.<br />
We thank ICMCB, Bordeaux, for providing the 10 nm CdS<br />
nanoparticles used in this study.<br />
SUPPORTING INFORMATION AVAILABLE<br />
Figure 1-ESI, Figure 2-ESI, and Figure 3-ESI. This material is<br />
available free of charge via the Internet at http://pubs.acs.org.<br />
Received for review May 12, 2010. Accepted July 8, 2010.<br />
AC101245H<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
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