Abstract：
Protein adducts are vital targets for exploring organophosphorus nerve agents (OPNAs) exposure and identification, that can be used to characterize the chemical burden and initiate chemical safety measures. However, the use of protein adducts as biomarkers of OPNA exposure has developed slowly. To further promote the development of biomarkers in chemical forensics, it is crucial to expand the range of modified peptides and active sites, and describe the characteristics of OPNA adducts at specific reaction sites. This study utilized multi-species and multi-source albumins as the protein targets. We identified 56 peptides in albumins from various species (including human, horse, rat and pig), that were modified by at least two OPNAs. Diverse modification characteristics were observed in response to certain agents: including (1) multiple sites on the same peptide modified by one or more agents, (2) different reactivities at the same site in homologous albumins, and (3) different preferences at the same active sites associated with differences in the biological matrix during exposure. Our studies provided an empirical reference with rationalized underpinnings supported by estimated conformation energetics through molecular modeling. We employed different peptide markers for detection of protein adducts, as (one would do) in forensic screening for identification and quantification of chemical damage. Three characteristic peptides were screened and analyzed in human albumin, including Y287ICENQDSISSK, K438VPQVS443TPTLVEVSR, and Y162LY164EIAR. Stable fragment ions with neutral loss were found from their tandem MS/MS spectra, which were used as characteristic ions for identification and extraction of modified peptides in enzymatic digestion mixtures. Coupling these observations with computer simulations, we found that the structural stability of albumin and albumin-adduct complexes (as well as the effective force that promotes stability of different adducts) changes in the interval before and after adduct formation. In pig albumin, five active peptides existed stably in vivo and in vitro. Most of them can be detected within 30 min after OPNA exposure, and the detection window can persist about half a month. These early findings provided the foundation and rationale for utilizing pig albumin as a sampling target for rapid analysis in future forensic work.
摘要：
蛋白质加合物是探索有机磷神经毒剂（OPNAs）暴露和鉴定的重要目标，可用于表征化学负荷并启动化学安全措施。然而,使用蛋白质加合物作为OPNA暴露的生物标志物进展缓慢。进一步推动生物标志物在化学法医学中的发展,扩大修饰肽和活性位点的范围至关重要，并描述了OPNA加合物在特定反应位点的特征。本研究利用多物种和多源白蛋白作为蛋白质靶标。我们鉴定了来自不同物种的白蛋白中的56种肽（包括人，马,老鼠和猪)，至少由两个OPNA修改。在响应某些试剂时观察到不同的修饰特征：包括（1）由一种或多种试剂修饰的同一肽上的多个位点，(2)同源白蛋白在同一位点的不同反应性，和(3)在暴露期间与生物基质的差异相关的相同活性位点处的不同偏好。我们的研究通过分子建模提供了由估计的构象能支持的合理化基础的经验参考。我们使用不同的肽标记来检测蛋白质加合物，就像(人们会做的)在法医筛查中识别和量化化学损害。在人白蛋白中筛选并分析了三种特征肽，包括Y287ICENQDSISSK,K438VPQVS443TPTLVEVSR,和Y162LY164EIAR。从串联MS/MS谱中发现了具有中性损失的稳定碎片离子，它们被用作鉴定和提取酶消化混合物中修饰肽的特征离子。将这些观察与计算机模拟相结合，我们发现白蛋白和白蛋白-加合物复合物的结构稳定性(以及促进不同加合物稳定性的有效作用力)在加合物形成前后的时间间隔内发生变化.在猪白蛋白中，五种活性肽在体内和体外稳定存在。它们中的大多数可以在OPNA暴露后30分钟内检测到，检测窗口可以持续约半个月。这些早期发现为将来在法医学工作中利用猪白蛋白作为快速分析的采样目标提供了基础和理由。
