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Figure 1.
Application of the quantitative MS method and structural
analysis for identification of the most susceptible Cys residue
of PTP1B to S-nitrosylation. A-D, recombinant PTP1B treated with
1 mm SNAP or 0.01 mm SNAP was subjected to differential isotope
labeling for quantitative MALDI-MS analysis as described in
Scheme 1. The full scan MALDI-MS profile (A) revealed three
pairs of cICAT-labeled tryptic peptides with a 9-Da difference,
which could be assigned to T4, T28, and T15, as shown in
expanded views (B-D), corresponding to the cICAT-labeled peptide
pairs containing Cys-32, Cys-215, or Cys-92, respectively. The
ratio of light/heavyc ICAT-labeled peak is shown below the
spectrum. E, the crystal of PTP1B was soaked with 1 mm SNAP at
room temperature for 20 min and subjected tox-ray
crystallography. The 2F[o] - 2F[c] electron density map showed a
mixture of reduced and S-nitrosylated states of Cys-215. Other
Cys residues (Cys-32, -92, -121, -226, and -231) remained in the
completely reduced form. Inset, the expended view of electron
density map illustrates the presence of an S-nitrosothiol form
of Cys-215.
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