FIGURE 10.
Reaction scheme of thiol-modifying reagents used for detection of unreacted free –SH group and MALDI-TOF MS analysis of cross-link formation in sQhpC. Reaction schemes for IAA (A) and NAM (D) and mass spectra for the sQhpC·QhpD (B and E) and sQhpCC7S·QhpD (C and F) complexes modified with IAA (B and C) or NAM (E and F) are shown. Note that sQhpC after undergoing the thioether bond formation would not be modified by these reagents. The reconstituted complexes (∼80 μm) before (blue) and after (red) the reaction with 1 mm DT and 1 mm SAM for 1 h were concentrated by precipitation with TCA, dissolved in 6 m urea in 50 mm potassium phosphate, incubated with 50 mm IAA or 0.5 mm NAM for 1 h, desalted with a C18 ZipTip pipette tip, and subjected to MALDI-TOF MS analysis. Theoretical m/z values (including St2-tag) ([M + H+]) are as follows: IAA-modified (uncross-linked) sQhpC, 8538.42; NAM-modified (uncross-linked) sQhpC, 8755.65; cross-linked sQhpC, 8479.35; uncross-linked sQhpCC7S, 8465.30.