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Using theoretical protein isotopic distributions to parse small-mass-difference post-translational interactions via mass spectrometry

机译：采用理论蛋白质同位素分布解析通过质谱小质量差的翻译后的相互作用

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Small-mass-difference modifications to proteins are obscured in mass spectrometry by the natural abundance of stable isotopes such as 13C that broaden the isotopic distribution of an intact protein. Using a ZipTip™ to remove salt from proteins in preparation for high-resolution mass spectrometry, the theoretical isotopic distribution intensities calculated from the protein’s empirical formula could be fit to experimentally acquired data and used to differentiate between multiple low-mass modifications to proteins. We could readily distinguish copper from zinc bound to a single-metal superoxide dismutase (SOD1) species; copper and zinc only differ by an average mass of 1.8 Daltons and have overlapping stable isotope patterns. In addition, proteins could be directly modified while bound to the ZipTip. For example, washing 11 mM S-methyl methanethiosulfonate over the ZipTip allowed the number of free cysteines on proteins to be detected as S-methyl adducts. Alternatively, washing with the sulfhydryl oxidant diamide could quickly reestablish disulfide bridges. Using these methods, we could resolve the relative contributions of copper and zinc binding, as well as disulfide reduction to intact SOD1 protein present from <100 µg of the lumbar spinal cord of a transgenic, SOD1 overexpressing mouse. Although techniques like ICP-MS can measure total metal in solution, this is the first method able to assess the metal-binding and sulfhydryl reduction of SOD1 at the individual subunit level and is applicable to many other proteins.
机译：质谱中的蛋白质小质量差异修饰因稳定同位素（例如 13 C）的天然丰度而变得模糊，这些同位素扩展了完整蛋白质的同位素分布。使用ZipTip™去除蛋白质中的盐以准备进行高分辨率质谱分析时，根据蛋白质的经验公式计算出的理论同位素分布强度可以适合实验获得的数据，并可以区分蛋白质的多种低质量修饰。我们可以很容易地将铜和锌结合到单金属超氧化物歧化酶（SOD1）上。铜和锌的平均质量差仅为1.8道尔顿，并且具有重叠的稳定同位素图谱。此外，与ZipTip结合时可以直接修饰蛋白质。例如，在ZipTip上洗涤11 mM S-甲硫基甲基磺酸盐可以检测到蛋白质上的游离半胱氨酸的数量为S-甲基加合物。或者，用巯基氧化剂二酰胺洗涤可快速重建二硫键。使用这些方法，我们可以解决铜和锌结合的相对贡献，以及二硫化物还原对完整的SOD1蛋白的作用，该蛋白来自＜100 µg过表达转基因SOD1小鼠的腰脊髓。尽管像ICP-MS这样的技术可以测量溶液中的总金属，但这是第一种能够评估单个亚基水平上SOD1的金属结合和巯基还原的方法，并且适用于许多其他蛋白质。

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