Zong Wansong, Wang Xiaoning, Yang Chuanxi, Du Yonggang, Sun Weijun, Xu Zhenzhen
College of Population, Resources and Environment, Shandong Normal University, Jinan, 250014, People's Republic of China.
Biomed Chromatogr. 2016 Jun;30(6):810-7. doi: 10.1002/bmc.3612. Epub 2015 Oct 22.
Research on biomarkers for protein oxidation might give insight into the mechanistic mode of oxidative stress. In the work present here, a novel pipeline was established to probe the oxidation mechanism of bovine hemoglobin (Hb) with its oxidation products serving as the biomarkers. Reactive oxygen species generated by irradiation were used to mimic oxidative stress conditions to oxidize Hb in bovine erythrocytes. After Hb extraction and digestion, oxidized peptides in the tryptic fragments were assigned by comparison with the extracted ion chromatography spectra of native peptide from the control sample. Subsequent tandem mass spectrometry analysis of these peptides proved that oxidation was limited to partially exposed amino acid residues (α-Phe36 , β-Met1 , β-Trp14 , for instance) in Hb. Quantitation analysis on these oxidized peptides showed that oxidation degrees of target sites had positive correlations with the extended oxidation dose and the oxidation processes were also controlled by residues types. Compared with the conventional protein carbonyl assay, the identified oxidized products were feasibility biomarkers for Hb oxidation, indicating that the proposed biomarker pipeline was suitable to provide specific and valid information for protein oxidation. Copyright © 2015 John Wiley & Sons, Ltd.
对蛋白质氧化生物标志物的研究可能有助于深入了解氧化应激的作用机制。在本文所述的研究中，建立了一种新型流程，以牛血红蛋白（Hb）的氧化产物作为生物标志物来探究其氧化机制。利用辐照产生的活性氧模拟氧化应激条件，氧化牛红细胞中的Hb。提取并消化Hb后，通过与对照样品中天然肽段的提取离子色谱图进行比较，确定胰蛋白酶水解片段中的氧化肽段。随后对这些肽段进行串联质谱分析，结果表明氧化仅限于Hb中部分暴露的氨基酸残基（例如α-Phe36、β-Met1、β-Trp14）。对这些氧化肽段的定量分析表明，靶位点的氧化程度与延长的氧化剂量呈正相关，且氧化过程也受残基类型的控制。与传统的蛋白质羰基检测法相比，所鉴定出的氧化产物是Hb氧化的可行性生物标志物，这表明所提出的生物标志物流程适合为蛋白质氧化提供具体且有效的信息。版权所有© 2015约翰威立父子有限公司。
