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首页> 外文OA文献 >Detection and enrichment of cytochrome P450s using bespoke affinity chromatography and proteomic techniques. Development of chemical immobilisation and novel affinity chromatography methods, with subsequent proteomic analysis, for the characterisation of cytochrome P450s important in cancer research.
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Detection and enrichment of cytochrome P450s using bespoke affinity chromatography and proteomic techniques. Development of chemical immobilisation and novel affinity chromatography methods, with subsequent proteomic analysis, for the characterisation of cytochrome P450s important in cancer research.

机译：使用定制亲和色谱和蛋白质组学技术检测和富集细胞色素P450。化学固定化和新型亲和色谱方法的发展，以及随后的蛋白质组学分析，用于表征在癌症研究中很重要的细胞色素P450。

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Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to ¿negatively¿ de-activate or ¿positively¿ activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects.udMethods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450¿s, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands.
机译：简介：细胞膜蛋白，例如细胞色素P450酶超家族（P450），在细胞生理中具有重要作用。 P450在代谢内源性分子以及代谢异源物质以排毒和排泄方面很重要。 P450也可能与癌症有关，因为它们可以“负向”停用或“正向”激活癌症治疗剂。鉴定在肿瘤部位上调高的特定P450可用于预测药物反应并制定靶向的癌症治疗方法以帮助减少全身性副作用。 ud方法：以前的富集策略无法分离出P450的全部补体超家族。为了开发用于P450的富集程序，开发了一种蛋白质组学策略，以便可以筛选化合物作为一般P450探针的有效性。创建了标准化的工作流程，包括亲和色谱，蛋白质浓缩/脱盐，十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和高效液相色谱-质谱（HPLC-MS）。将具有已知P450抑制作用的酮康唑类似物和2-EN类似物固定在固体支持物上，以与固定的组胺进行比较。利用辅因子的去除，竞争性洗脱和探针二硫键的DTT裂解来洗脱结合的蛋白质。结果/讨论：抑制珠结合了多种蛋白质，包括P450，其中一些通过辅因子去除被洗脱，一些通过竞争性洗脱。通过优化缓冲液条件和固相支持物可以提高结合的特异性，但是不能完全消除非特异性结合。使用一种或多种配体从加标样品中提取所有人类P450，从更复杂的小鼠肝脏样品中回收18 P450。

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    Bateson Hannah;

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