Chem Res Toxicol. 2019 Oct 21;32(10):2086-2094. doi: 10.1021/acs.chemrestox.9b00272. Epub 2019 Sep 13.
-Formyl-lysine (FLys) is an abundant and lasting protein adduct formed when formaldehyde generated by nitrosative/oxidative stress and inflammation reacts with lysine residues. It is believed that the post-translational -formylation of lysine is associated with a variety of pathological processes and human diseases. Thus, FLys may serve well as a dosimetric biomarker for exposure to formaldehyde and other oxidative stress-inducing toxicants. However, since current methods for FLys determination are tedious and time-consuming, we developed and validated an aqueous normal phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with isotope-dilution method for the rigorous quantification of FLys with enhanced sensitivity and selectivity. After validating the accuracy and precision of the method with a synthetic peptide containing FLys, the method was applied to quantitate the concentration-dependent formation of FLys in cells exposed to formaldehyde and Fe-EDTA, an OH radical-mediated oxidant. The study reveals formaldehyde and Fe-EDTA produced FLys at a frequency of 20.2 and 4.1 per 10 lysine per mM, respectively, after correcting for losses during protein digestion steps. The study was further extended to quantitate the concentration-dependent formation of FLys in aristolochic acid I (AA-I) exposed cells and rat tissues. This study demonstrates for the first time that AA-I exposure induces time- and dose-dependent formation of FLys in cellular proteins. Furthermore, results show AA-I exposure leads to organotropic -formylation of lysine, with elevated levels of FLys detectable in the kidney, which is the one of the tumor targeting organs of AAs. Previous studies have also revealed AA exposure induced renal interstitial fibrosis in both laboratory rodents and humans, by a yet to be determined molecular mechanism. These data shed light on the potential caustative role of -formylation in the pathophysiology of AA nephrotoxicity and carcinogenicity.
甲酰赖氨酸（FLys）是一种丰富且持久的蛋白质加合物，当由氧化应激和炎症产生的甲醛与赖氨酸残基反应时形成。据信，赖氨酸的翻译后 - 甲酰化与各种病理过程和人类疾病有关。因此，FLys 可以作为暴露于甲醛和其他氧化应激诱导的毒物的剂量测定生物标志物。然而，由于目前测定 FLys 的方法繁琐且耗时，因此我们开发并验证了一种水相正相液相色谱-串联质谱（LC-MS/MS）与同位素稀释法相结合的方法，该方法具有增强的灵敏度和选择性，可严格定量 FLys。在用含有 FLys 的合成肽验证该方法的准确性和精密度后，该方法被应用于定量测定暴露于甲醛和 Fe-EDTA（一种 OH 自由基介导的氧化剂）的细胞中 FLys 的浓度依赖性形成。该研究揭示了甲醛和 Fe-EDTA 在蛋白质消化步骤中损失校正后，分别以每 10 个赖氨酸每 mM 产生 20.2 和 4.1 个 FLys 的频率产生 FLys。该研究进一步扩展到定量测定 AA-I 暴露的细胞和大鼠组织中 FLys 的浓度依赖性形成。这项研究首次表明，AA-I 暴露会诱导细胞蛋白中 FLys 的时间和剂量依赖性形成。此外，结果表明 AA-I 暴露导致赖氨酸的器官特异性 - 甲酰化，可在肾脏中检测到升高的 FLys 水平，肾脏是 AA 的肿瘤靶向器官之一。先前的研究还揭示了 AA 暴露通过尚未确定的分子机制在实验室啮齿动物和人类中引起肾间质纤维化。这些数据揭示了 - 甲酰化在 AA 肾毒性和致癌性的病理生理学中的潜在因果作用。
