Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 6. List of ions used for the determination of pig kidney MT sequences. (a) MT-1g, (b) MT-1h, (c) N-Ac-MT-1g, (d) MT-1a and MT-2a,
(e) N-Ac-MT-1h, (f) N-Ac-MT-2a, (g) N-Ac-MT-1a, (h) N-Ac-MT-2a. Bold characters highlight residues where modifications are observed.
no mass fragment specific to MT-1e in MS/MS spectra, its
presence or absence cannot be unambiguously confirmed. Even
if present, its contribution to the MTs pool would be very low. By
selecting appropriate and narrow retention time ranges in the MS/
MS chromatogram, y ions detected covered 74% and 87% of the
MT-1a and MT-2a amino acid sequence (Figure 6d) confirming
the identification of Cd7MT-1a and Cd7MT-2a.
An amino acid sequence of MT-2a without the N-terminal was
observed for the peak eluting at 40.5 min (Figure 6f). The mass
difference with regard to MT-2a isoforms indicated N-acetylation
on methionine residue leading to the identification of N-Ac-MT-
2a. This was also confirmed by two peaks of the MT2a specific
fragments (acetylated and non acetylated) in Figure 3-ESI e. The
sequence of the MT eluting at 41 min suggests strongly its
6956 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
identification as N-Ac-MT-1a (δ -0.3 ppm). Indeed, the presence
of the sequence fragment 39 Ala -Gly-Cys-Ala 42 and the overall
sequence coverage of 95% supported this identification.
The molecular masses of the remaining four apo-MTs in Table
2 did not correspond to any of the reported pig MTs subisoforms.
The 42-Da difference between the forms eluting at 33.8 and 37.4
min (peaks 1 and 3) and between the forms eluting at 35 and
38.5 min (peaks 2 and 5) indicated two pairs of acetylated and
non-acetylated isoforms. The amino acid sequence of the MT
subisoform eluting at 33.8 min (5940.124 Da, Figure 6a) was found
to be as a mixture of MT-1a and MT-1e as m/z 1091.924 and m/z
940.374 (charge +2) ions were observed for, respectively, Ala39
and Lys43 residues and matched the theoretical y ions (m/z
1091.929 and m/z 940.376 (charge +2). Furthermore, a y ion at