Graduate School of Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory, 1060 Commerce Park, Oak Ridge, Tennessee 37830, USA.
J Proteome Res. 2013 Jul 5;12(7):3307-16. doi: 10.1021/pr400141p. Epub 2013 Jun 19.
Redox proteomics has yielded molecular insight into diseases of protein dysfunction attributable to oxidative stress, underscoring the need for robust detection of protein oxidation products. Additionally, oxidative protein surface mapping techniques utilize hydroxyl radicals to gain structural insight about solvent exposure. Interpretation of tandem mass spectral data is a critical challenge for such investigations, because reactive oxygen species target a wide breadth of amino acids. Additionally, oxidized peptides may be generated in a wide range of abundances since the reactivity of hydroxyl radicals with different amino acids spans 3 orders of magnitude. Taken together, these attributes of oxidative footprinting pose both experimental and computational challenges to detecting oxidized peptides that are naturally less abundant than their unoxidized counterparts. In this study, model proteins were oxidized electrochemically and analyzed at both the intact protein and peptide levels. A multidimensional chromatographic strategy was utilized to expand the dynamic range of oxidized peptide measurements. Peptide mass spectral data were searched by the "hybrid" software packages Inspect and Byonic, which incorporate de novo elements of spectral interpretation into a database search. This dynamic search capacity accommodates the challenge of searching for more than 40 oxidative mass shifts that can occur in a staggering variety of possible combinatorial occurrences. A prevailing set of oxidized residues was identified with this comparative approach, and evaluation of these sites was informed by solvent accessible surface area gleaned through molecular dynamics simulations. Along with increased levels of oxidation around highly reactive "hotspot" sites as expected, the enhanced sensitivity of these measurements uncovered a surprising level of oxidation on less reactive residues.
氧化还原蛋白质组学为蛋白质功能障碍疾病（归因于氧化应激）提供了分子见解，强调了需要稳健地检测蛋白质氧化产物。此外，氧化蛋白质表面图谱技术利用羟基自由基获得有关溶剂暴露的结构见解。对于此类研究，串联质谱数据的解释是一个关键挑战，因为活性氧物种针对广泛的氨基酸。此外，由于羟基自由基与不同氨基酸的反应性跨越 3 个数量级，因此氧化肽可能以广泛的丰度产生。总之，氧化足迹的这些特性对检测自然丰度低于其未氧化对应物的氧化肽既具有实验性又具有计算性挑战。在这项研究中，模型蛋白通过电化学氧化进行了分析，并且在完整蛋白和肽水平上进行了分析。利用多维色谱策略扩展了氧化肽测量的动态范围。肽质荷比谱数据通过“混合”软件包 Inspect 和 Byonic 进行搜索，该软件包将光谱解释的从头元素纳入数据库搜索中。这种动态搜索能力适应了搜索 40 多个氧化质量位移的挑战，这些位移可能以令人惊讶的多种可能组合发生。通过这种比较方法确定了一组占主导地位的氧化残基，并且通过分子动力学模拟获得的可及表面积对这些位点的评估提供了信息。除了在高度反应性的“热点”位点周围的氧化水平增加外，这些测量的增强敏感性还揭示了在反应性较低的残基上令人惊讶的氧化水平。
