Using isotopically-coded hydrogen peroxide as a surface modification reagent for the structural characterization of prion protein aggregates.

The conversion of the cellular prion protein (PrPC) into aggregated ß-oligomeric (PrPß) and fibril (PrPSc) 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 PrPC and PrPβ. 16O and 18O hydrogen peroxide were used to oxidize methionine and tryptophan residues in PrPC and PrPβ, 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 16O2 and H2 18O2), the PrPC and PrPβ forms of the protein were then combined, digested with trypsin, and analysed by LC-MS. The 18O/16O signal intensity ratios were used to determine the relative levels of oxidation of specific amino acids in the PrPC and PrPβ 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 PrPC involved in the formation of PrPβ 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. Biological significance: Surface modification using isotopically-coded hydrogen peroxide has allowed quantitative comparison of the exposure of methionine and tryptophan residues in PrPC and PrPß 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? [ABSTRACT FROM AUTHOR]