Using microwave-assisted metal-catalyzed oxidation reactions and mass spectrometry to increase the rate at which the copper-binding sites of a protein are determined
Using microwave-assisted metal-catalyzed oxidation reactions and mass spectrometry to increase the rate at which the copper-binding sites of a protein are determined
复制标题
DOI：
10.1021/ac0502551
复制
发表时间：
2005-07-15
影响因子：
7.4
通讯作者：
Vachet, RW
中科院分区：
文献类型：
--
作者：
Bridgewater, JD;Vachet, RW
受控微波辐射已被用于加速金属催化氧化（MCO）反应，该反应可位点特异性氧化金属蛋白中与Cu结合的氨基酸。当与质谱相结合时，这些MCO反应提供了一种测定cu蛋白结合位点的灵敏方法。在这项工作中，我们证明微波辐射可以使Cu/Zn超氧化物歧化酶的这些位点特异性氧化反应的速率增加至少15倍。通过选择合适的微波功率，可以在加速氧化反应的同时避免改变蛋白质的结构。通过测量不同肽段氧化程度与微波功率的关系，可以很容易地确定维持蛋白质结构完整性所需的最大微波功率。这样的确定需要多次测量，从而限制了该方法的总体吞吐量；然而，这些微波辅助反应确实为研究cu结合位点的动态变化提供了很好的时间分辨率。
Controlled microwave irradiation has been used to accelerate metal-catalyzed oxidation (MCO) reactions that site-specifically oxidize the amino acids bound to Cu in a metalloprotein. When combined with mass spectrometry, these MCO reactions provide a sensitive method for determining Cu-protein binding sites. In this work, we demonstrate that microwave irradiation can increase the rate of these site-specific oxidation reactions for Cu/Zn superoxide dismutase by at least 15-fold. By choosing the appropriate microwave power, changes to the protein's structure can be avoided while still accelerating the oxidation reactions. The maximum microwave power suitable for maintaining the protein's structural integrity can be readily determined by measuring the oxidation extent of different peptide fragments as a function of microwave power. Such determinations require several measurements and thus limit the overall throughput of this approach; however, these microwave-assisted reactions do provide good time resolution for studying dynamic changes to Cu-binding sites.