Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry
Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry
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DOI：
10.1080/09553000110094805
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发表时间：
2002-02-01
影响因子：
2.6
通讯作者：
Chance, MR
中科院分区：
文献类型：
--
作者：
Kiselar, JG;Maleknia, SD;Chance, MR
报道了一种将同步辐射和质谱学相结合来探测蛋白质结构的新方法。利用同步加速器光对蛋白质溶液进行辐射分解时产生的羟基在毫秒时间尺度上修饰氨基酸侧链。这会导致形成稳定的氧化产物，其中活性残留物的氧化程度受到其侧链对主体溶剂的可访问性的影响。已发现芳香族和含硫残基优先反应，这与以前的多肽研究一致。用串联质谱仪确定了氧化的位置。根据质谱仪检测到的修饰和未修饰的多肽离子的相对比例，测量了许多蛋白水解肽的氧化速率随暴露时间的变化。氧化速率与天然蛋白质结构中残基侧链对溶剂的可及性的理论测量密切相关。这种方法可以区分溶菌酶62和123位的色氨酸残基侧链和所有其他色氨酸残基的相对可及性，以及34位的苯丙氨酸和3和38位的苯丙氨酸残基的氧化速率。
A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the structure of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions using synchrotron light modify amino acid side-chains on millisecond timescales. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side-chains to the bulk solvent. The aromatic and sulphur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for a number of proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates correlate closely with a theoretical measure of the accessibility of residue side-chains to the solvent in the native protein structure. This approach can distinguish the relative accessibility of the tryptophan residue side-chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues, and phenylalanine at position 34 from phenylalanine residues at positions 3 and 38 based upon their rates of oxidation.