Battelle at the Centers for Disease Control and Prevention , Atlanta, Georgia 30341, United States.
Centers for Disease Control and Prevention , National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Georgia 30341, United States.
Anal Chem. 2017 Feb 7;89(3):1955-1964. doi: 10.1021/acs.analchem.6b04441. Epub 2017 Jan 24.
In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with <1 ppm and tied them to signature product ions with <5 ppm deviation from their chemical formulas. This high mass accuracy data from precursor and product ions, collected over 23 independent immunoprecipitation preparations, established method operating limits. QC data and experiments with 14 synthetic reference peptides indicated that reliable qualitative identification of biomarkers was possible for analytes >15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents.
在这项研究中，开发了一种依赖数据的高分辨率串联质谱（ddHRMS/MS）方法，能够检测到所有与人体丁酰胆碱酯酶（BChE）结合的有机磷神经毒剂（OPNA）加合物。在暴露事件发生后，用 BChE 特异性抗体从血液中免疫沉淀，并使用胃蛋白酶消化，产生含有 OPNA 加合物的九肽。这种胃蛋白酶 BChE 九肽（FGES*AGAAS）的特征产物离子为广泛筛选 OPNA 暴露提供了一种途径。在 HRMS 仪器上采用这种方法可以识别具有高精度质量的生物标志物，包括未知物。使用一组暴露于 OPNA 的混合人血清作为质量控制（QC）材料，该方法成功地鉴定了前体离子，其精度<1ppm，并将其与特征产物离子联系起来，其偏差<5ppm 偏离其化学式。这种来自前体和产物离子的高精度质量数据，在 23 个独立的免疫沉淀制备物上收集，建立了方法的操作限制。QC 数据和用 14 种合成参考肽进行的实验表明，对于>15ng/mL 的分析物，可靠地定性鉴定生物标志物是可能的。该方法应用于 96 个未暴露血清样本的便利集和 80 个用 OPNA 处理的盲样集。在便利样本集中未观察到 OPNA 生物标志物，在盲样集中未观察到假阳性或假阴性鉴定。盲样血清集中所有>15ng/mL 的生物标志物均被正确鉴定。这是首次报道一种 ddHRMS/MS 方法，该方法能够补充现有的定量方法，并适合于识别未知有机磷试剂的暴露。
