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Figure 2.
Protective effect of Cys S-nitrosylation on preventing PTP1B
from further irreversible oxidation. A, recombinant PTP1B was
pretreated with 1 mm N-acetylpencillamine or SNAP for 20 min,
followed by 1 mm H[2]O[2] for 10 min, and then digested by
trypsin in solution. The tryptic peptides were subjected to
LC-nESI-MS analysis. T28 carrying the Cys-215 in Cys-SH,
Cys-SO[2]H, Cys-SO[3]H, and Cys-SNO forms were first identified
by manually examining the nESI-MS profile at the expected
retention time. For a semiquantitative assessment, the extracted
ion chromatograms for the respective signals are plotted in A
without normalization or correcting for nESI-MS response factor.
The amount of the irreversibly oxidized SO[2]H/SO[3]H form
(eluting at 17.7-18.5 min) elicited by 1 mm H[2]O[2] is clearly
reduced to basal level in SNAP-pretreated sample, compared with
N-acetylpencillamine-pretreated sample. The corresponding
nESI-MS profiles for this time point are shown in B, where the
signals identified as the irreversibly oxidized T28 (m/z 736.4
and 741.7) are detectable at increasing intensity when PTP1B was
treated with increasing concentration of H[2]O[2] but not when
it was first S-nitrosylated by SNAP. C, recombinant PTP1B,
either directly exposed to H[2]O[2], SNAP, or GSNO or pretreated
with SNAP or GSNO followed by H[2]O[2] treatment, was subjected
to immunoblotting with an anti-oxidized PTP active site
(anti-oxi-PTP) antibody (top) or an anti-PTP1B antibody (FG6)
(bottom). The effect of SNAP or GSNO on inhibiting the level of
H[2]O[2]-induced irreversible oxidation of PTP1B was observed in
three independent experiments.
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