A novel approach for quantitation of N-terminal valine adducts

描述（由申请人提供）：人类不断暴露于各种混合物，如烟草烟雾，汽车尾气和其他环境污染物，含有数千种化合物，包括许多已知的致癌物。反应性代谢物与DNA和蛋白质形成稳定加合物的共价结合被认为是暴露与致癌之间的因果关系。DNA和蛋白质加合物是公认的内有效剂量的生物标记物，是基于科学的风险评估的组成部分。然而，由于技术上的限制，无法同时对不同加合物的谱进行全面评估。因此，大多数研究都集中在确定丰富的个体或选择少数加合物上。这些研究对个别致癌物的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造成的风险所需的准确和全面的数据。因此，我们的长期目标是利用血红蛋白加合物作为替代生物标志物，通过开发一种敏感和特异性的方法来对广谱活性化合物或其代谢物进行定量分析，从而克服这一限制。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），包括胰蛋白酶水解和在LC-MS/MS定量之前通过加合物特异性免疫亲和层析（IAC）富集样品。我们在此建议重新设计IAC富集程序，以便同时定量广谱的n端缬氨酸加合物。我们基于这样的假设，即广泛的烷基化n端肽可以通过使用特异性针对目标肽的c端提出的抗体来富集。我们的初步研究表明，这种新设计适用于生物监测。为了验证这一假设，我们将（目标1）证明所提出的多加合物监测方法的原理证明，该方法采用烷基化肽标准和珠蛋白，在体外用已知形成n端缬氨酸加合物的烷基化剂处理；（目标2）建立多加合物监测方法在体内生物监测中的适用性，使用暴露于各种烷基化剂浓度低于吸烟和职业环境水平的小鼠和大鼠的珠蛋白。我们建议在小鼠、大鼠和人类中建立这种方法，以实现转化研究。这种新颖的重新设计能够同时确定几种致癌物的内部剂量，这将有可能(a)更好地了解混合物中单个化合物的行为，(b)产生更全面的暴露数据，以准确评估暴露于混合物的风险。所提出的方法可以很容易地扩展到包括其他感兴趣的加合物，并适应于研究除n端缬氨酸以外的其他位点的烷基化，提供一种通用的分析方法来理解活性物质如何与大分子相互作用以显示其不利影响。

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.