LC-MS/MS Methods for Analysis of Hemoglogbin Adducts

描述(由申请人提供)： 血红蛋白与活性化学物质或其代谢物之间形成的加合物提供了一种监测长期接触化学物质的手段。然而，通常用于测量这些加合物的方法对一种或最多几种化学物质是选择性的，很难执行，通常需要用气相色谱和质谱仪来进行加合物分析。这项研究的目标将是开发可用于研究的方法 接触多种试剂，而不是选择性地针对单一分析物。一种广泛使用的分析与血红蛋白N-末端缬氨酸残基反应生成的加合物的方法是改进的Edman降解法。 在这项技术中，利用负离子化学电离质谱仪对缬氨酸加合物的五氟苯基硫代海因衍生物进行了定量。我们实验室进行的初步研究表明，改进的Edman降解释放的丙烯酰胺和缩水甘油酰胺加合物的分析可以通过苯硫代海因衍生物的液质联用(LC-MS)完成，在三重四重质谱仪上进行多反应监测。固相萃取减少了 样品准备时间。我们的新分析方法在样品准备和分析时间方面都节省了相当多的时间。 拟议研究的目标将是使该方法适用于在同一样品中对来自各种不同关注化学品的一些加合物进行定量，并使用选择性较低的方法推导出分析珠蛋白中存在的加合物的方法。用改进的Edman降解法结合离子陷阱MS上的LC-MS分析来检测与Valine的加合物，将为鉴定存在的加合物提供必要的灵敏度。对珠蛋白加合物形成的定性评价 蛋白质消化和质谱学主要是在体外进行的，在体内的应用数量有限。 这些研究的另一个目的是将蛋白质消化和串联质谱学应用于体内加合物形成的评估，并评估该方法用于定性评估暴露的敏感性。然而，测量加合物仍然是一种相对较低的吞吐量方法，不适用于涉及数千个样本的大规模流行病学研究，尽管这比目前可用的要快。

DESCRIPTION (provided by applicant): Adducts formed between hemoglobin and reactive chemicals or their metabolites provide a means of monitoring of the long-term exposure to chemicals. However, the methods that are generally used for measuring these adducts are selective for one or at most several chemicals, are difficult to perform, and typically involve gas chromatography with mass spectrometry for adduct analysis. The objective of this study will be to develop methods that can be used to study exposure to a variety of agents, rather than selectively targeted to a single analyte. A widely used method for analysis of adducts formed on reaction with the N-terminal valine residue of hemoglobin is the modified Edman degradation. In this technique, the pentafluorophenylthiohydantoin derivative of the valine adduct is quantitated by negative ion chemical ionization mass spectrometry. Preliminary studies conducted in our laboratory indicate that the analysis of hemoglobin adducts of acrylamide and glycidamide released by a modified Edman degradation can be accomplished by liquid chromatraphy-mass spectrometry (LC-MS) of phenylthiohydantoin derivatives, using multiple reaction monitoring on a triple quadruple mass spectrometer. Solid phase extraction has reduced sample preparation time. Our new method of analysis provides considerable time savings both in sample preparation and analysis time. Objectives of the proposed research will be to adapt the methodology to the quantitation of a number of adducts derived from a variety of different chemicals of concern in the same samples, and to derive methods for analysis of adducts that are present in globin using less selective methods. The detection of adducts with valine using the modified Edman degradation coupled with LC-MS analysis on an ion trap MS will provide the necessary sensitivity for identification of the adducts present. The qualitative evaluation of adduct formation in globin by protein digestion and mass spectrometry has been carried out primarily in vitro, with applications in vivo in a limited number of cases. A further aim of these studies will be to apply protein digestion and tandem mass spectrometry to the evaluation of adduct formation in vivo, and to evaluate the sensitivity of this approach for the qualitative evaluation of exposure. However, measuring adducts is still relatively low throughput methodology that is not amenable to large-scale epidemiological studies involving thousands of samples, although this is faster than what is currently available.