Reactive Nitrogen Oxide Species-Induced Post-Translational Modifications in Human Hemoglobin and the Association with Cigarette Smoking
Reactive Nitrogen Oxide Species-Induced Post-Translational Modifications in Human Hemoglobin and the Association with Cigarette Smoking
复制标题
DOI：
10.1021/ac301597r
复制
发表时间：
2012-09-18
影响因子：
7.4
通讯作者：
Chen, Yu-Chin
中科院分区：
文献类型：
--
作者：
Chen, Hauh-Jyun Candy;Chen, Yu-Chin
一氧化氮（NO）是正常生理所必需的，但在炎症过程中NO的过量产生会损害邻近组织。活性氮氧化物（RNOx），包括过氧亚硝酸盐（ONOO-），是强大的硝化剂。生物蛋白质硝化涉及几种疾病状态，包括炎性疾病，并且通过在发炎组织中检测3-硝基酪氨酸（3 NT）来证明。在这项研究中，我们确定了过氧亚硝基诱导的翻译后修饰（PTM）在人类血红蛋白的准确质量测量以及MS 2和MS 3光谱。鉴定了Tyr-24、Tyr-42（α-球蛋白）和Tyr-130（β-球蛋白）上的硝化以及Tyr-24（α-球蛋白）上的亚硝化。还表征了所有三个甲硫氨酸残基（α-Met-32、α-Met-76和β-Met-SS）氧化为亚砜，以及半胱氨酸氧化（测定为α-Cys-104上的亚磺酸和α-Cys-104、β-Cys-93和β-Cys-112上的磺酸）。在从人血液中新鲜分离的血红蛋白中检测这些修饰，并且在选择反应监测（SRM）模式下通过纳流液相色谱-纳喷雾电离串联质谱（nanoLC-NSI/MS/MS）相对于参考肽半定量修饰的程度。结果显示，吸烟与α-Tyr-24和α-Tyr-42的酪氨酸硝化程度之间存在统计学显著正相关。据我们所知，这是第一份关于鉴定和定量人血血红蛋白中多种PTM以及特定含3 NT肽与吸烟相关性的报告。这种高灵敏度和特异性的测定仅需要从一滴血液（类似于10 μ L）中分离的血红蛋白。因此，测量血红蛋白中的这些PTM可能是可行的，用于评估体内硝化应激。
Nitric oxide (NO) is essential for normal physiology, but excessive production of NO during inflammatory processes can damage the neighboring tissues. Reactive nitrogen oxide species (RNOx), including peroxynitrite (ONOO-), are powerful nitrating agents. Biological protein nitration is involved in several disease states, including inflammatory diseases, and it is evident by detection of 3-nitrotyrosine (3NT) in inflamed tissues. In this study, we identified peroxynitrite-induced post-translational modifications (PTMs) in human hemoglobin by accurate mass measurement as well as by the MS2 and MS3 spectra. Nitration on Tyr-24, Tyr-42 (alpha-globin), and Tyr-130 (beta-globin) as well as nitrosation on Tyr-24 (alpha-globin) were identified. Also characterized were oxidation of all three methionine residues, alpha-Met-32, alpha-Met-76, and beta-Met-SS to the sulfoxide, as well as cysteine oxidation determined as sulfinic acid on alpha-Cys-104 and sulfonic acid on alpha-Cys-104, beta-Cys-93, and beta-Cys-112. These modifications are detected in hemoglobin freshly isolated from human blood and the extents of modifications were semiquantified relative to the reference peptides by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the selected reaction monitoring (SRM) mode. The results showed a statistically significant positive correlation between cigarette smoking and the extents of tyrosine nitration at alpha-Tyr-24 and at alpha-Tyr-42. To our knowledge, this is the first report on identification and quantification of multiple PTMs in hemoglobin from human blood and association of a specific 3NT-containing peptide with cigarette smoking. This highly sensitive and specific assay only requires hemoglobin isolated from one drop (similar to 10 mu L) of blood. Thus, measurement of these PTMs in hemoglobin might be feasible for assessing nitrative stress in vivo.