Domingues Pedro, Fonseca Conceição, Reis Ana, Domingues M Rosário M
Mass Spectrometry Centre, Department of Chemistry, University of Aveiro, Aveiro, Portugal.
Biomed Chromatogr. 2012 Jan;26(1):51-60. doi: 10.1002/bmc.1624. Epub 2011 Mar 23.
Free radical species are generally short-lived due to their high reactivity and thus direct measurement and identification are often impossible. In this study we used a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), to trap radical intermediates formed during the oxidation of isomeric dipeptides tyrosine-leucine (Tyr-Leu) and leucine-tyrosine (Leu-Tyr), induced by the hydroxyl radical. To investigate the influence of the amino acid position in the peptide chain on the oxidation and free radical generation, the spin adducts were characterized using LC-MS and MS(n) . We detected carbon and oxygen DMPO adducts and adducts bearing two DMPO, which were analyzed by MS(n) . Both alkoxyl and peroxyl radicals were identified. Radical intermediates were localized in Tyr during oxidation of Tyr-Leu, while radicals were identified in Leu and Tyr during oxidation of Leu-Tyr. DMPO adducts of acyl radical species formed from cleavage of the peptide backbone, promoted by the alkoxyl radical in α carbon of the N-terminal amino acid were observed. The results show that the amino acid position has an influence in the oxidation process, at least on small peptides, and that the α carbon of the N-terminal amino acid is more vulnerable to the attack of the electrophilic hydroxyl radical.
自由基物种通常因其高反应活性而寿命短暂,因此往往无法直接进行测量和鉴定。在本研究中,我们使用了一种自旋捕获剂5,5-二甲基-1-吡咯啉-N-氧化物(DMPO)来捕获由羟基自由基诱导的同分异构二肽酪氨酸-亮氨酸(Tyr-Leu)和亮氨酸-酪氨酸(Leu-Tyr)氧化过程中形成的自由基中间体。为了研究肽链中氨基酸位置对氧化和自由基生成的影响,使用液相色谱-质谱联用仪(LC-MS)和多级质谱(MS(n))对自旋加合物进行了表征。我们检测到了碳和氧的DMPO加合物以及带有两个DMPO的加合物,并通过MS(n)对其进行了分析。同时鉴定出了烷氧基和过氧自由基。在Tyr-Leu氧化过程中,自由基中间体定位于酪氨酸(Tyr)上,而在Leu-Tyr氧化过程中,在亮氨酸(Leu)和酪氨酸(Tyr)中均鉴定出了自由基。观察到由N端氨基酸α碳上的烷氧基自由基促进肽主链裂解形成的酰基自由基物种的DMPO加合物。结果表明,氨基酸位置对氧化过程有影响,至少对小肽是这样,并且N端氨基酸的α碳更容易受到亲电羟基自由基的攻击。