Application of Mass Spectrometry in Pharmacology

在这一竞争性更新中，要求支持我们继续研究获得性耐药机制。我们建议全面研究当癌细胞对不同的化疗药物产生耐药性时，蛋白质丰度和结构发生的各种变化。基于质谱的蛋白质组学策略将用于比较MCF-7人乳腺癌细胞中蛋白质的丰度和结构，这些细胞通过暴露于依托oposide， mitoxantrone和melphalan来选择三种亚群。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进行深思熟虑的考虑，将为获得性耐药性的机制和转化细胞的超敏感性提供新的见解。可能会发现新的治疗靶点。这并不是一个高通量的项目，相反，将对细胞器和亚组分进行重叠和重复的比较，以便识别和阐明由于突变或翻译后修饰引起的结构变化。在一个平行的和相关的研究中，将比较放射性同位素标记的melphalan在易感和耐药细胞、亚组分和细胞器中的蛋白质烷基化模式。相关性，或缺乏相关性，将寻求在比较研究中定义的烷基化目标的性质，烷基化目标的变化和细胞生物化学的变化之间的相关性。

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.