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 2Fo - 2Fc 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.