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品或其代谢物之间形成的加合物提供了一种监测长期暴露于化学品的手段。然而，通常用于测量这些加合物的方法对一种或至多几种化学品是选择性的，难以执行，并且通常涉及气相色谱法和质谱法用于加合物分析。本研究的目的是开发可用于研究 暴露于各种试剂，而不是选择性地靶向单一分析物。一种广泛用于分析与血红蛋白的N-末端缬氨酸残基反应形成的加合物的方法是改良的Edman降解。 在该技术中，缬氨酸加合物的五氟苯硫代乙内酰脲衍生物通过负离子化学电离质谱法定量。在我们的实验室进行的初步研究表明，血红蛋白加合物的丙烯酰胺和缩水甘油酰胺释放的修改埃德曼降解的分析可以完成苯硫代海因衍生物的液相色谱-质谱法（LC-MS），使用多反应监测的三重四重质谱仪。固相萃取法降低了 样品制备时间。我们的新分析方法在样品制备和分析时间方面都节省了大量时间。 拟议的研究的目标将是调整的方法，以定量的一些加合物来自各种不同的化学品的关注，在相同的样品，并得出的加合物，存在于珠蛋白使用选择性较低的方法进行分析的方法。使用改进的Edman降解结合离子阱MS上的LC-MS分析检测与缬氨酸的加合物，将为鉴定存在的加合物提供必要的灵敏度。用免疫荧光法定性评价珠蛋白加合物的形成 蛋白质消化和质谱分析主要在体外进行，在有限数量的情况下在体内应用。 这些研究的另一个目的是将蛋白质消化和串联质谱法应用于体内加合物形成的评价，并评价这种方法用于定性评价暴露的灵敏度。然而，测量加合物仍然是相对低通量的方法，不适合涉及数千个样品的大规模流行病学研究，尽管这比目前可用的方法更快。

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.