title: Using isotopically-coded hydrogen peroxide as a surface modification reagent for the structural characterization of prion protein aggregates.
abstract: The conversion of the cellular prion protein (PrP(C)) into aggregated ß-oligomeric (PrP(ß)) and fibril (PrP(Sc)) forms is the central element in the development of prion diseases. Here we report the first use of isotopically-coded hydrogen peroxide surface modification combined with mass spectrometry (MS) for the differential characterization of PrP(C) and PrP(β). (16)O and (18)O hydrogen peroxide were used to oxidize methionine and tryptophan residues in PrP(C) and PrP(β), allowing for the relative quantitation of the extent of modification of each form of the prion protein. After modification with either light or heavy forms of hydrogen peroxide (H2(16)O2 and H2(18)O2), the PrP(C) and PrP(β) forms of the protein were then combined, digested with trypsin, and analysed by LC-MS. The (18)O/(16)O signal intensity ratios were used to determine the relative levels of oxidation of specific amino acids in the PrP(C) and PrP(β) forms. Using this approach we have detected several residues that are differentially-oxidized between the native and β-oligomeric prion forms, allowing determination of the regions of PrP(C) involved in the formation of PrP(β) aggregates. Modification of these residues in the β-oligomeric form is compatible with a flip of the β1-H1-β2 loop away from amphipathic helices 2 and 3 during conversion.
  
  
  CONCLUSIONS
  Surface modification using isotopically-coded hydrogen peroxide has allowed quantitative comparison of the exposure of methionine and tryptophan residues in PrP(C) and PrP(ß) forms of prion protein. Detected changes in surface exposure of a number of residues have indicated portions of the PrP structure which undergo conformational transition upon conversion. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?
authors: University of Victoria, Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada.
authors: Jason J
authors: JJ
authors: Serpa
authors: University of Victoria, Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada.
authors: Karl A T
authors: KA
authors: Makepeace
authors: University of Victoria, Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada.
authors: Tristan H
authors: TH
authors: Borchers
authors: Departments of Biological Sciences and Computing Science, University of Alberta, Edmonton, Alberta, T6G 2E8, Canada.
authors: David S
authors: DS
authors: Wishart
authors: University of Victoria, Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada.
authors: Evgeniy V
authors: EV
authors: Petrotchenko
authors: University of Victoria, Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada; University of Victoria, Department of Biochemistry & Microbiology, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada. Electronic address: christoph@proteincentre.com.
authors: Christoph H
authors: CH
authors: Borchers
mesh_terms: D006861:Hydrogen Peroxide / Q000737:chemistry*; D007553:Isotope Labeling; D008715:Methionine / Q000032:analysis / Q000737:chemistry; D010100:Oxygen; D010103:Oxygen Isotopes; D018621:PrPC Proteins / Q000737:chemistry*; D018620:PrPSc Proteins / Q000737:chemistry*; D011328:Prions / Q000737:chemistry; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D014364:Tryptophan / Q000032:analysis / Q000737:chemistry
chemical_list: D010103:Oxygen Isotopes; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011328:Prions; D014364:Tryptophan; D008715:Methionine; D006861:Hydrogen Peroxide; D010100:Oxygen
keywords: Chemical surface modification; Mass spectrometry; Oxidative labeling; Prion aggregate structure; Stable isotope labeled hydrogen peroxide; Structural proteomics