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Molecular & Cellular Proteomics 8:706-719, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Sulfonation and Phosphorylation of Regions of the Dioxin Receptor Susceptible to Methionine Modifications^*,S

Keyur A. Dave^,, Fiona Whelan^¶, Colleen Bindloss^¶, Sebastian G. B. Furness^||, Anne Chapman-Smith^¶, Murray L. Whitelaw^¶ and Jeffrey J. Gorman^,**

From the Protein Discovery Centre, Queensland Institute of Medical Research, P. O. Royal Brisbane Hospital, Herston, Queensland 4029, Australia, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia, ^¶ School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia 5005, Australia, and ^|| Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia

Tagged murine dioxin receptor was purified from mammalian cells, digested with trypsin, and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. Several chromatographically distinct semitryptic peptides matching two regions spanning residues Glu⁴⁰⁹–Arg⁴²⁴ and Ser⁵⁴⁷–Arg⁵⁵⁵ of the dioxin receptor were revealed by de novo sequencing. Methionine residues at 418 and 548 were detected in these peptides as either unmodified or modified by moieties of 16 (oxidation) or 57 amu (S-carboxamidomethylation) or in a form corresponding to degradative removal of 105 amu from the S-carboxamidomethylated methionine. MS/MS spectra revealed that the peptides containing modified methionine residues also existed in forms with a modification of +80 amu on serine residues 411, 415, and 547. The MS/MS spectra of these peptide ions also revealed diagnostic neutral loss fragment ions of 64, 98, and/or 80 amu, and in some instances combinations of these neutral losses were apparent. Taken together, these data indicated that serines 411 and 547 of the dioxin receptor were sulfonated and serine 415 was phosphorylated. Separate digests of the dioxin receptor were prepared in H₂¹⁶O and H₂¹⁸O, and enzymatic dephosphorylation was subsequently performed on the H₂¹⁶O digest only. The digests were mixed in equal proportions and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. This strategy confirmed assignment of sulfonation as the cause of the +80-amu modifications on serines 411 and 547 and phosphorylation as the predominant cause of the +80-amu modification of serine 415. The relative quantitation of phosphorylation and sulfonation enabled by this differential phosphatase strategy also suggested the presence of sulfonation on a serine other than residue 411 within the sequence spanning Glu⁴⁰⁹–Arg⁴²⁴. This represents the first description of post-translational sulfonation sites and identification of a new phosphorylation site of the latent dioxin receptor. Furthermore this is only the second report of serine sulfonation of eukaryotic proteins. Mutagenesis studies are underway to assess the functional consequences of these modifications.

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