Lee et al. 10.1073/pnas.0702081104. |
Fig. 6. S-cysteinylation of OhrR. OhrR (300 nM) was oxidized with CHP (3 mM) in the absence (trace 2) or presence of various concentrations of free Cys as described in Fig. 1A. After 30-min incubation, proteins were recovered by TCA precipitation, dissolved in 2% acetic acid and 50% methanol, and analyzed by ESI-MS. Right shows the deconvoluted spectra derived from multiply charged ions (Left).
Fig. 7. Analysis of modified T3 peptides by ESI-MS/MS. (A) b and y fragment ions and their expected masses for the T3 tryptic peptide.(B) T3 without modification. Note that 10 mM DTT was added to prevent adventitious oxidation. The appearance of the y9 ion (1,059.5 Da) is indicative of fully reduced peptide. (C) T3+119 peptide (S-cysteinylated T3) contains both the y8 and y9 + 119 ions, indicative of S-thiolation on Cys15. (D) T3+305 peptide (S-glutathionylated peptide). A characteristic feature of S-glutathionylated peptides is a major product ion generated by neutral loss of 129 Da by MS/MS(MS2). (E) Further analysis (MS3) of the T3+305-129 peptide. The presence of y and b ions of T3 confirms that the loss of 129 Da occurred at a glutathione moiety linked to Cys15. (F) MS/MS of IA modified T3 peptide. (G) MS/MS of CoA-modified T3 peptide. The loss of 410, 428, and 508 Da from the parent ion is characteristic of CoA-modified peptides. The doubly charged parent ions are indicated by blue diamonds.
1. Fuangthong M, Helmann JD (2002) Proc Natl Acad Sci USA 99:6690-6695.
Fig. 9. Formation of intersubunit disulfide bond by partially oxidized OhrR. (A) OhrR was desalted to remove DTT and analyzed by SDS/PAGE. In the presence of SDS, a small amount of protein dimer is formed under denaturing conditions. This dimer is not detected in samples pretreated with either IA or AMS. This indicates that disulfide bond formation occurs in the unfolded state upon reaction of oxidized OhrR (Cys15-SOH) with unoxidized OhrR (Cys15-SH). (B). The amount of the protein disulfide does not increase over time in reactions to which no CHP has been added. (C-H) These six images correspond to the entire SDS/PAGE gels for Figs. 2D and 3 A-D, respectively. Disulfide-linked dimers form spontaneously when samples containing partially oxidized OhrR (containing both SH and SOH forms of Cys15) are concentrated by TCA precipitation and suspended in nonreducing SDS sample buffer. Thus, maximal dimer formation occurred ~5-10 min after 300 nM CHP addition (C) or ~30 sec after 3 mM CHP addition (E). This length of time corresponds to half-maximal oxidation as judged by AMS modification. As noted previously (1), native OhrR does not form disulfide-linked dimers upon oxidation, nor was any disulfide-linked dimer detected at longer time points (over 10 min), after reduction with DTT (F) or in samples containing Cys (D and G).
1. Fuangthong M, Helmann JD (2002) Proc Natl Acad Sci USA 99:6690-6695.
Fig. 10. Time course of sulfenamide formation. OhrR (300 nM) was oxidized by 3 mM CHP in the absence of thiol (same conditions as Fig. 1A, Fig. 3A, or SI Fig. 6). At the indicated time, proteins were recovered by TCA precipitation and analyzed by ESI-MS as for Fig. 3E. (Left) The spectra of multiply charged ions with m/z values and corresponding total charges. (Center) The deconvoluted spectra corresponding to OhrR monomer. (Right) Only for + 1 min and + 2 min samples are spectra shown corresponding to OhrR dimer (as visualized also in SI Fig. 9E). The dimeric species is detected in the primary data as species in between the major peaks for the monomeric species. For example, the 920.2-Da m/z peak corresponds to a dimer with a +37 net charge and is intermediate in m/z values between the +18 and +19 charged monomers. Note that the dimeric species in this experiment result from the concentration of the protein by TCA precipitation at a time when there is a mixture of protein sulfenic acid and protein thiol species (see SI Fig. 9).
Fig. 11. ESI-MS analysis of oxidized OhrR (in the absence of Cys). (A and B) OhrR (300 nM) was either untreated (A) or treated with 3 mM CHP for 30 min (B) as for Fig. 3E. Protein was recovered by TCA precipitation and digested with trypsin before analysis by ESI-MS. The arrows indicate the [T3 + 2H+] parental ions. (C and D) Spectra obtained after CID are presented. The b and y series fragment ions (see SI Fig. 7A) are shown. For both peaks the y8 ion is unmodified, whereas the y9 and b9 ions (see Inset) are decreased in mass by 2 Da. These results localized the mass loss to the Cys15 residue.
Fig. 12. The T3+396 Da peak is modified on Cys15. Analysis of the m/z = 2,053 peak by MS/MS indicates that this peptide is derived from the T3 peptide and modified on Cys15. (A) Expected b and y ions from the T3 peptide. (B) Collision-induced dissociation (CID) MS/MS analysis of doubly charged m/z = 1,027 peak by ESI-MS. (C) Postsource decay (PSD) MS/MS analysis of m/z = 2,053 peak by MALDI-TOF MS. Samples were prepared from B. subtilis by IP followed by SDS/PAGE and in-gel trypsin digestion as in Fig. 4B.
Fig. 13. Detection of CoASH-modified OhrR by MALDI-TOF (negative ion mode). OhrR-FLAG proteins were recovered from B. subtilis as described in Fig. 4, except that analysis was done in negative ion mode. (A) In the untreated sample, the T3 peptide was detected in both the IA-modified (T3+57 Da; white triangle) and apparently unmodified (black triangle) forms. (B) Sample from CHP-treated cells revealed the presence of the T3+396 Da peak (dark blue triangle; only detected in B) and a small peak corresponding to the CoASH mixed disulfide (light blue triangle; only detected in B). In addition, there is a significant amount of sulfonic acid (T3+49 Da; red triangle) detected in this ionization mode. The S-cysteinylated peptide is not detected in this analysis (yellow triangle). (C) The sample from B was incubated with 10 mM DTT for 10 min before MALDI-TOF MS analysis. Note the loss of both the CoASH and + 396-Da S-thiolated peaks. There is also a decrease in the signal intensity of the sulfonic acid peak, suggesting that this oxidized peak accumulates, in part, from oxidation during sample preparation and analysis by MS. Note that the T3 peptide exhibited a m/z value of 1,653.7, which corresponds to 1-Da loss from expected m/z value of [T3-H]- (m/z = 1,654.8).