Site-specific amide hydrogen/deuterium exchange in E-coli thioredoxins measured by electrospray ionization mass spectrometry

Mass spectrometry as an analytical tool to study protein folding and struct ure by hydrogen/deuterium exchange is a relatively new approach. In this st udy, site-specific amide deuterium content was measured in oxidized and red uced E coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D2O phosphate-buffered solut ion (pH 5.7). The deuterium levels correlated well with reported NMR-determ ined H/D exchange rate constants. The deuterium measured by y(n) ions, howe ver, showed much less reliable correlation with rate exchange data. In gene ral, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deute rium levels, Most amide sites in the two protein forms showed similar deute rium levels consistent with the expected similarity of their structures, bu t there were some differences. The turn consisting of residues 18-22 in par ticular showed more variability in deuterium content consistent with report ed structural differences in the two forms. The deuterium uptake by thiored oxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroet hyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-5 3 and Thr-54, showed slightly elevated deuterium levels, suggesting that st ructural changes had occurred from alkylation of the protein at Cys-32. It is concluded that b(n) ions are reliable for determining the extent of site -specific amide hydrogen isotope exchange and that mass spectrometry is use ful as a complementary technique to NMR and other analytical methods for pr obing regional structural characteristics of proteins.