Application of Mass Spectrometry in Pharmacology

在这一竞争性更新中，需要得到支持，以继续我们对获得性耐药性机制的研究。我们提出了一个全面的研究，当癌细胞对不同的化疗药物产生耐药性时，癌细胞中发生的蛋白质丰度和结构的变化。基于质谱的蛋白质组学策略将用于比较MCF-7人乳腺癌细胞与通过暴露于依托泊苷、米托蒽醌和美法仑选择的三个亚系中蛋白质的丰度和结构。预计将发现比以前认识到的更大的蛋白质阵列被改变。我们还期望，对观察到的各种变化的深思熟虑将为获得性耐药机制和转化细胞的超敏感性提供新的见解。可以确定新的治疗靶点。这不是一个高通量的项目，而是将进行细胞器和亚组分的重叠和重复比较，以允许识别和阐明由于突变或翻译后修饰引起的结构变化。在一项平行和相关的研究中，将在敏感和耐药细胞、亚组分和细胞器中比较放射性同位素标记的美法仑的蛋白质烷基化模式。将在比较研究中定义的烷基化靶标的性质、烷基化靶标的变化和细胞生物化学的变化之间寻找相关性或缺乏相关性。

Support is requested in this competitive renewal to continue our studies of mechanisms of acquired drug resistance. We propose a comprehensive study of the kinds of changes in protein abundance and structures that occur in cancer cells when they become resistant to different chemotherapeutic agents. Mass spectrometry-based proteomics strategies will be used to compare the abundances and structures of proteins in MCF-7 human breast cancer cells with three sublines selected by exposure to etoposide, mitoxantrone and melphalan. It is expected that a much larger array of proteins will be found to be altered than has been previously recognized. We also expect that thoughtful consideration of the kinds of changes observed will provide new insights into mechanisms of acquired resistance, and into the hyper-sensitivity of transformed cells. New therapeutic targets may be identified. This is not envisioned as a high throughput project, but rather, overlapping and reiterative comparisons of organelles and sub-fractions will be made to allow structural changes due to mutations or post-translational modifications to be recognized and elucidated. In a parallel and related investigation, patterns of protein alkylation by radio-isotope labeled melphalan will be compared in susceptible and resistant cells, sub- fractions and organelles. Correlations, or the lack thereof, will be sought between the nature of the alkytation targets, changes in the alkylation targets, and changes in cell biochemistry defined in the comparative study.