Serpa Jason J, Makepeace Karl A T, Borchers Tristan H, Wishart David S, Petrotchenko Evgeniy V, Borchers Christoph H
University of Victoria, Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada.
Departments of Biological Sciences and Computing Science, University of Alberta, Edmonton, Alberta, T6G 2E8, Canada.
J Proteomics. 2014 Apr 4;100:160-6. doi: 10.1016/j.jprot.2013.11.020. Epub 2013 Dec 3.
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.
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?
细胞朊蛋白（PrP(C)）转化为聚集的β-寡聚体（PrP(β)）和纤维状（PrP(Sc)）形式是朊病毒疾病发展的核心要素。在此，我们报告首次使用同位素编码的过氧化氢表面修饰结合质谱（MS）对PrP(C)和PrP(β)进行差异表征。使用(16)O和(18)O过氧化氢氧化PrP(C)和PrP(β)中的甲硫氨酸和色氨酸残基，从而对朊蛋白每种形式的修饰程度进行相对定量。在用轻或重形式的过氧化氢（H2(16)O2和H2(18)O2）修饰后，将蛋白质的PrP(C)和PrP(β)形式混合，用胰蛋白酶消化，然后通过液相色谱-质谱分析。(18)O/(16)O信号强度比用于确定PrP(C)和PrP(β)形式中特定氨基酸的相对氧化水平。使用这种方法，我们检测到了天然和β-寡聚体朊病毒形式之间差异氧化的几个残基，从而确定了参与PrP(β)聚集体形成的PrP(C)区域。β-寡聚体形式中这些残基的修饰与转化过程中β1-H1-β2环从两亲性螺旋2和3翻转开来相一致。
使用同位素编码的过氧化氢进行表面修饰能够对朊蛋白的PrP(C)和PrP(β)形式中甲硫氨酸和色氨酸残基的暴露情况进行定量比较。检测到的多个残基表面暴露的变化表明了PrP结构中在转化时发生构象转变的部分。本文是名为《蛋白质组学能否填补基因组学与表型之间的空白？》特刊的一部分。
