Rapid Method for Quantifying the Extent of Methionine Oxidation in Intact Calmodulin
We have developed a method for rapidly quantifying the extent to which the functionally important Met144 and Met145 residues near the C-terminus of calmodulin (CaM) are converted to the corresponding sulfoxides, Met(O). The method utilizes a whole protein collision induced dissociation (CID) approach on an electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer. Using standards of CaM oxidized by hydrogen peroxide (H2O2) or peroxynitrite (ONOO-), we demonstrated that CID fragmentation of the protein ions resulted in a series of C-terminal singly charged y1?y15 ions. Fragments larger than y4 exhibited mass shifts of +16 or +32 Da, corresponding to oxidation of one or two methionines, respectively. To assess the extent of oxidative modification for Met144 and Met145 to Met(O), we averaged the ratio of intensities for yn, yn +16, and yn +32 ions, where n = 6?9. By alternating MS and CID scans at low and high collision energies, this technique allowed us to rapidly determine both the distribution of intact CaM oxiforms and the extent of oxidative modification in the C-terminal region of the protein in a single run. We have applied the method to studies of the repair of fully oxidized CaM by methionine sulfoxide reductases (MsrA and MsrB), which normally function in concert to reduce the S and R stereoisomers of methionine sulfoxide. We found that repair of Met(O)144 and Met(O)145 did not go to completion, but was more efficient than average Met repair. Absence of complete repair is consistent with previous studies showing that accumulation of methionine sulfoxide in CaM can occur during aging.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 15020830
- Report Number(s):
- PNWD-SA-7078; TRN: US200521%%365
- Journal Information:
- Journal of the American Society for Mass Spectrometry, Vol. 16, Issue 9
- Country of Publication:
- United States
- Language:
- English
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