A novel approach for quantitation of N-terminal valine adducts

描述(申请人提供)：人类经常接触到各种混合物，如烟草烟雾、汽车尾气和其他环境污染物，其中包含数千种化合物，包括许多已知的致癌物质。反应性代谢产物与DNA和蛋白质的共价结合以及稳定加合物的形成被认为是接触和致癌之间的因果联系。DNA和蛋白质加合物是公认的内部有效剂量的生物标志物，是以科学为基础的风险评估的组成部分。然而，技术上的限制阻碍了对不同加合物的板谱同时进行全面评估。因此，大多数研究都集中在对大量个体或少数加合物的测定上。这些研究对个体致癌物的新陈代谢产生了有价值的见解。不幸的是，它们不足以提供评估接触混合物所构成的风险所需的准确和全面的数据。因此，我们的长期目标是通过开发一种灵敏和特异的方法来克服这一限制，以血红蛋白加合物作为替代生物标志物来定量分析广谱的活性化合物或其代谢物。我们最近建立了一种免疫亲和液相色谱-串联质谱法(LC-MS/MS)，该方法包括胰酶水解和样品通过加合物特异的免疫亲和层析(IAC)富集，然后用LC-MS/MS进行定量。我们建议在此重新设计IAC富集法，以便能够同时定量N-末端valine加合物。我们基于这样一个假设，即通过使用针对靶肽C末端产生的抗体，可以丰富广谱的烷基化N末端多肽。我们的初步研究表明，这种新设计适合于生物监测。为了验证这一假说，我们将(目标1)证明拟议的多加合物监测方法的原理证明，该方法使用烷基化肽标准，并且在体外用已知的烷基化剂处理的珠蛋白可形成N-末端缬氨酸加合物；以及(目标2)建立多加合物监测方法的适宜性，使用在吸烟期间和在职业环境中暴露于低于经验浓度的各种烷化剂的小鼠和大鼠的珠蛋白来进行体内生物监测。我们建议为小鼠、大鼠和人类建立这种方法，以实现转译研究。这一新的重新设计能够同时确定几种致癌物质的内部剂量，这将使人们能够(A)更好地了解混合物中个别化合物的行为，以及(B)生成准确评估接触混合物的风险所需的更全面的暴露数据。所提出的方法可以很容易地扩展到包括更多感兴趣的加合物，并适合于研究N-末端valine以外的位置的烷基化反应，为了解反应剂如何与大分子相互作用展示其不利影响提供了一般的分析方法。 与公共健康相关：人类经常接触到各种混合物，如烟草烟雾、汽车尾气和其他环境污染物，其中包含数千种化合物，包括许多已知的致癌物。人们认为，接触具有基因毒性的混合物与产生不良健康影响之间的因果联系，是致癌物或其代谢物与DNA和形成所谓加合物的蛋白质的结合。然而，技术上的限制阻碍了对多个加入物的同时评估。因此，我们建议开发一种方法，使其能够同时确定多种加合物的存在和数量，以便获得评估接触混合物对人类健康构成的风险所需的准确信息。

DESCRIPTION (provided by applicant): Humans are constantly exposed to various mixtures, such as tobacco smoke, auto exhaust, and other environmental pollutants, containing several thousand compounds, including many known carcinogens. Covalent binding of reactive metabolites to DNA and proteins with the formation of stable adducts is believed to be the causal link between exposure and carcinogenesis. DNA and protein adducts are well established biomarkers for the internal effective dose and are an integral part of science-based risk assessment. Technical limitations, however, have prevented comprehensive assessment of a board spectrum of different adducts simultaneously. Consequently, most studies have focused on determination of abundant individual or a select few adducts. These studies have produced valuable insights into metabolism of individual carcinogens. Unfortunately they are insufficient in providing accurate and comprehensive data needed for assessment of the risk posed by exposure to mixtures. Thus, our long-term goal is to overcome this limitation by developing a sensitive and specific method for quantitative profiling of a broad spectrum of reactive compounds or their metabolites using hemoglobin adducts as surrogate biomarkers. We recently established an immunoaffinity liquid chromatography-tandem mass spectrometry method (LC-MS/MS) consisting of trypsin hydrolysis and sample enrichment by an adduct-specific immunoaffinity chromatography (IAC) prior to quantitation by LC-MS/MS. We propose herein to redesign the IAC enrichment procedure to enable simultaneous quantitation of a broad spectrum of N-terminal valine adducts. We base this redesign on the hypothesis that a broad spectrum of alkylated N-terminal peptides can be enriched with the use of antibodies raised specifically against the C-terminus of the target peptide. Our preliminary studies suggest that this new design is suitable for bio-monitoring. To test this hypothesis, we will (Aim 1) demonstrate proof-of-principle of the proposed multi-adduct-monitoring method with alkylated peptide standards and globin treated in vitro with alkylating agents known to form N-terminal valine adducts; and (Aim 2) establish the suitability of the multi-adduct-monitoring method for in vivo bio-monitoring using globin from mice and rats exposed to various alkylating agents at concentrations lower than experienced during smoking and at levels of occupational settings. We propose to establish this methodology for mice, rats, and humans to enable translational research. This novel redesign enabling determination of the internal doses of several carcinogens simultaneously will make it possible to (a) better understand the behavior of individual compounds in mixtures, and (b) generate more comprehensive exposure data needed for accurate risk assessment of exposure to mixtures. The proposed methodology can easily be extended to include additional adducts of interest and adapted to investigate alkylation at sites other than the N-terminal valine, providing a general analysis approach to understanding how reactive agents interact with macromolecules to exhibit their adverse effects. PUBLIC HEALTH RELEVANCE: Humans are constantly exposed to various mixtures, such as tobacco smoke, auto exhaust, and other environmental pollutants, containing several thousand compounds, including many known carcinogens. The causal link between exposure to genotoxic mixtures and the development of adverse health effects is believed to be the binding of carcinogens or their metabolites to DNA and proteins forming so called adducts. Technical limitations, however, have prevented the assessment of a multiple adducts simultaneously. Therefore, we propose to develop a method to make it possible to determine the presence and number of multiple adducts simultaneously in order to obtain accurate information needed to assess the risks to human health posed by exposure to mixtures.