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Simultaneous Mass Spectrometric Analysis of Methylated and Ethylated Peptides in Human Hemoglobin: Correlation with Cigarette Smoking
Chemical Research in Toxicology ( IF 4.1 ) Pub Date : 2017-10-12 00:00:00 , DOI: 10.1021/acs.chemrestox.7b00234
Hauh-Jyun Candy Chen,Sun Wai Ip,Fu-Di Lin

Alkylating agents contained in cigarettes smoke might be related to cancer development. Post-translational protein methylation and ethylation may cause alteration of protein functions. Human hemoglobin (Hb) has been a target for molecular dosimetry because of its easy accessibility. The goal of this study is to investigate the relationship between the levels of methylation and ethylation at specific sites of Hb with smoking. Because of the low extent of modification of Hb isolated from blood, the methylation and ethylation sites were identified in Hb incubated with a methylating agent (methyl methanesulfonate, MMS) and ethylating agent (ethyl methanesulfonate, EMS), respectively, by accurate mass measurements. After trypsin digestion, the modification sites were identified by nanoflow LC–nanospray ionization coupled with high-resolution mass spectrometry. The selected reaction monitoring mode was used to quantify the relative extent of methylation and ethylation in human Hb incubated with MMS and EMS, respectively. Methylation occurred at 9 sites, including 1V, 20H, 50H, 72H of α-globin and 1V, 26E, 66K, 77H, 93C of β-globin. Ethylation was detected at 11 sites, including 1V, 16K, 50H, 72H, 87H of α-globin and 1V, 17K, 66K, 77H, 92H, 93C of β-globin. The relative extents of methylation and ethylation were measured in blood samples from 13 smokers and 13 nonsmokers. No statistically significant difference was found in the methylated peptides. On the other hand, the extents of ethylation at α-terminal Val, α-His-50, α-His-87, β-terminal Val, β-His-77, and β-Cys-93 in Hb were significantly higher in smokers than in nonsmokers (p < 0.05). Furthermore, the relative extents of ethylation at these sites were statistically significantly correlated with the number of cigarettes smoked per day. Therefore, this assay, which requires as little as one drop of blood, should be helpful in measuring Hb ethylation as a potential biomarker for assessing the exposure to cigarette smoking.

中文翻译:

人体血红蛋白中甲基化和乙基化肽的同时质谱分析:与吸烟的相关性

卷烟烟雾中所含的烷基化剂可能与癌症的发展有关。翻译后蛋白质的甲基化和乙基化可能导致蛋白质功能的改变。人类血红蛋白(Hb)由于易于获取而成为分子剂量测定的目标。这项研究的目的是调查吸烟中Hb特定部位的甲基化和乙基化水平之间的关系。由于从血液中分离出的Hb修饰程度较低,因此通过精确的质量测量分别在与甲基化剂(甲烷磺酸甲酯,MMS)和乙基化剂(甲烷磺酸乙酯,EMS)孵育的Hb中鉴定出甲基化和乙基化位点。胰蛋白酶消化后,修饰位点通过纳流液相色谱-纳米喷雾电离结合高分辨率质谱法鉴定。所选的反应监测模式用于量化分别与MMS和EMS孵育的人血红蛋白中甲基化和乙基化的相对程度。甲基化发生在9个位点,包括1 V,20 H,50 H,72 H的α-珠蛋白和1 V,26 E,66 K,77 H,93 C的β-珠蛋白。在11个位点检测到乙氧基化,包括1 V,16 K,50 H,72 H,87 Hα-珠蛋白和1 V,17 K,66 K,77 H,92 H,93β-珠蛋白的C。在来自13位吸烟者和13位非吸烟者的血液样本中测量了甲基化和乙基化的相对程度。在甲基化的肽中未发现统计学上的显着差异。另一方面,Hb中α端Val,α-His-50,α-His-87,β端Val,β-His-77和β-Cys-93的乙基化程度明显更高。吸烟者比不吸烟者(p <0.05)。此外,在这些位置上乙基化的相对程度在统计学上与每天吸烟的数量显着相关。因此,这种只需少一滴血液的测定方法就有助于测量Hb乙基化水平,以此作为评估吸烟量的潜在生物标记。
更新日期:2017-10-12
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