Johnson Research Foundation, Department of Biochemistry and Biophysics, and Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
Proc Natl Acad Sci U S A. 2013 Oct 8;110(41):16438-43. doi: 10.1073/pnas.1315532110. Epub 2013 Sep 9.
Hydrogen exchange technology provides a uniquely powerful instrument for measuring protein structural and biophysical properties, quantitatively and in a nonperturbing way, and determining how these properties are implemented to produce protein function. A developing hydrogen exchange-mass spectrometry method (HX MS) is able to analyze large biologically important protein systems while requiring only minuscule amounts of experimental material. The major remaining deficiency of the HX MS method is the inability to deconvolve HX results to individual amino acid residue resolution. To pursue this goal we used an iterative optimization program (HDsite) that integrates recent progress in multiple peptide acquisition together with previously unexamined isotopic envelope-shape information and a site-resolved back-exchange correction. To test this approach, residue-resolved HX rates computed from HX MS data were compared with extensive HX NMR measurements, and analogous comparisons were made in simulation trials. These tests found excellent agreement and revealed the important computational determinants.
氢交换技术为测量蛋白质结构和生物物理特性提供了一种独特而强大的工具，能够定量且非干扰地测量这些特性，并确定这些特性是如何实现蛋白质功能的。一种正在发展的氢交换-质谱法（HX-MS）能够分析大型生物重要的蛋白质系统，同时只需要非常少量的实验材料。HX-MS 方法的主要剩余缺陷是无法将 HX 结果解析为单个氨基酸残基分辨率。为了实现这一目标，我们使用了一个迭代优化程序（HDsite），该程序整合了最近在多肽获取方面的进展，以及以前未检查过的同位素包络形状信息和具有分辨率的反向交换校正。为了测试这种方法，从 HX-MS 数据计算得到的残基分辨 HX 速率与广泛的 HX NMR 测量进行了比较，并在模拟试验中进行了类似的比较。这些测试发现了极好的一致性，并揭示了重要的计算决定因素。
