Untargeted adductomics of Cys34 modifications to human serum albumin in newborn dried blood spots.
Untargeted adductomics of Cys34 modifications to human serum albumin in newborn dried blood spots.
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新生儿干血斑中 Cys34 修饰对人血清白蛋白的非靶向加合组学。
DOI：
10.1007/s00216-019-01675-8
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发表时间：
2019
影响因子：
4.3
通讯作者：
Rappaport,Stephen
中科院分区：
文献类型：
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作者：
Yano,Yukiko;Grigoryan,Hasmik;Schiffman,Courtney;Edmands,William;Petrick,Lauren;Hall,Katie;Whitehead,Todd;Metayer,Catherine;Dudoit,Sandrine;Rappaport,Stephen
饮食中化学物质的代谢、暴露于异种生物、微生物群和生活方式因素（如吸烟、饮酒）产生亲电试剂，与循环蛋白中的亲核位点反应，特别是人血清白蛋白（HSA）的Cys34。为了发现子宫内暴露导致的潜在危险因素，我们正在调查新生儿干血斑（DBS）中反映妊娠最后一个月全身暴露的HSA-Cys34加合物。工作流程包括从DBS中提取蛋白质，测量血红蛋白（Hb）以使血容量归一化，通过沉淀Hb和其他干扰蛋白加入甲醇以丰富HSA，用胰蛋白酶消化，通过纳米流液相色谱-高分辨率质谱法检测HSA- cys34加合物。作为原理证明，我们将该方法应用于49例从母亲在怀孕期间积极吸烟或不吸烟的新生儿中收集的存档DBS。检测到26种HSA-Cys34加合物，包括Cys34氧化产物、低分子量硫醇混合二硫化物（如半胱氨酸、同型半胱氨酸、谷胱甘肽、半胱氨酸甘氨酸）和其他修饰物。用一种新的方法（“scone”）对数据进行归一化，以消除由HSA消化、血容量、DBS年龄、质谱分析和批次效应引起的不必要的技术变化。使用线性和非线性模型的集合，发现氰化物的Cys34加合物在吸烟和不吸烟母亲的新生儿中具有一致的区别，平均倍数变化（吸烟/不吸烟）为1.31。这些结果表明，DBS内收组学适用于研究子宫内暴露于可能影响生命后期疾病风险的活性化学物质和代谢物。
Metabolism of chemicals from the diet, exposures to xenobiotics, the microbiome, and lifestyle factors (e.g., smoking, alcohol intake) produce electrophiles that react with nucleophilic sites in circulating proteins, notably Cys34 of human serum albumin (HSA). To discover potential risk factors resulting from in utero exposures, we are investigating HSA-Cys34 adducts in archived newborn dried blood spots (DBS) that reflect systemic exposures during the last month of gestation. The workflow includes extraction of proteins from DBS, measurement of hemoglobin (Hb) to normalize for blood volume, addition of methanol to enrich HSA by precipitation of Hb and other interfering proteins, digestion with trypsin, and detection of HSA-Cys34 adducts via nanoflow liquid chromatography-high-resolution mass spectrometry. As proof-of-principle, we applied the method to 49 archived DBS collected from newborns whose mothers either actively smoked during pregnancy or were nonsmokers. Twenty-six HSA-Cys34 adducts were detected, including Cys34 oxidation products, mixed disulfides with low molecular weight thiols (e.g., cysteine, homocysteine, glutathione, cysteinylglycine), and other modifications. Data were normalized with a novel method (“scone”) to remove unwanted technical variation arising from HSA digestion, blood volume, DBS age, mass spectrometry analysis, and batch effects. Using an ensemble of linear and nonlinear models, the Cys34 adduct of cyanide was found to consistently discriminate between newborns of smoking and nonsmoking mothers with a mean fold change (smoking/nonsmoking) of 1.31. These results indicate that DBS adductomics is suitable for investigating in utero exposures to reactive chemicals and metabolites that may influence disease risks later in life.