Department of Chemistry and Biochemistry, National Chung Cheng University, Ming-Hsiung, Chia-Yi, Taiwan.
Anal Chem. 2012 Sep 18;84(18):7881-90. doi: 10.1021/ac301597r. Epub 2012 Sep 7.
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 MS(2) and MS(3) spectra. Nitration on Tyr-24, Tyr-42 (α-globin), and Tyr-130 (β-globin) as well as nitrosation on Tyr-24 (α-globin) were identified. Also characterized were oxidation of all three methionine residues, α-Met-32, α-Met-76, and β-Met-55 to the sulfoxide, as well as cysteine oxidation determined as sulfinic acid on α-Cys-104 and sulfonic acid on α-Cys-104, β-Cys-93, and β-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 α-Tyr-24 and at α-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 (∼10 μL) of blood. Thus, measurement of these PTMs in hemoglobin might be feasible for assessing nitrative stress in vivo.
一氧化氮（NO）对正常生理至关重要，但在炎症过程中过量产生的 NO 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。生物蛋白硝化参与了几种疾病状态，包括炎症性疾病，并且可以通过在发炎组织中检测到 3-硝基酪氨酸（3NT）来证明。在这项研究中，我们通过精确质量测量以及 MS(2)和 MS(3)谱鉴定了过氧亚硝酸盐诱导的人血红蛋白的翻译后修饰（PTMs）。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。还鉴定了三个甲硫氨酸残基α-Met-32、α-Met-76 和 β-Met-55 的氧化，以及α-Cys-104 上的半胱氨酸氧化确定为亚磺酸和α-Cys-104、β-Cys-93 和 β-Cys-112 上的磺酸。这些修饰物是在新鲜分离自人血液的血红蛋白中检测到的，并且通过纳米流液相色谱-纳喷雾电离串联质谱法（nanoLC-NSI/MS/MS）在选定反应监测（SRM）模式下，相对于参考肽，通过半定量测定了修饰物的程度。结果表明，吸烟与 α-Tyr-24 和 α-Tyr-42 处酪氨酸硝化的程度之间存在统计学上显著的正相关。据我们所知，这是首次报道从人血液中血红蛋白中鉴定和定量多种 PTMs，并将含有特定 3NT 的肽与吸烟联系起来。这种高度敏感和特异的测定法仅需要从一滴（约 10 μL）血液中分离的血红蛋白。因此，血红蛋白中这些 PTMs 的测量可能可用于评估体内硝化应激。
