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Rapid method for quantifying the extent of methionine oxidation in intact calmodulin

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Journal of the American Society for Mass Spectrometry

Abstract

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 y n , y n + 16, and y n + 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 (Gao, J.; Yin, D.; Yao, Y.; Williams, T. D.; Squier, T. C. Biochemistry 1998, 37, 9536–9548).

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Authors and Affiliations

  1. Mass Spectrometry Laboratory, University of Kansas, 2010 Malott Hall, 1251 Wescoe Hall Drive, 66045-7582, Lawrence, KS, USA

    Nadezhda A. Galeva, S. Wynn Esch & Todd D. Williams

  2. Pacific Northwest National Laboratory, Richland, Washington, USA

    Lye Meng Markille & Thomas C. Squier

Authors
  1. Nadezhda A. Galeva
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  2. S. Wynn Esch
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  3. Todd D. Williams
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  4. Lye Meng Markille
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  5. Thomas C. Squier
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Correspondence to Todd D. Williams.

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Published online July 14, 2005

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Galeva, N.A., Esch, S.W., Williams, T.D. et al. Rapid method for quantifying the extent of methionine oxidation in intact calmodulin. J Am Soc Mass Spectrom 16, 1470–1480 (2005). https://doi.org/10.1016/j.jasms.2005.04.009

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  • DOI: https://doi.org/10.1016/j.jasms.2005.04.009

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