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The development and application of methods for activity-based protein profiling.

机译：基于活动的蛋白质谱分析方法的开发和应用。

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摘要

Considering that proteins mediate nearly all biochemical events underlying cell and organismal physiology and pathophysiology, the need to develop general methods to measure levels and activities of these biomolecules directly in cell, and tissue proteomes is apparent. The ability to profile classes of proteins based on activity would greatly accelerate assignment of protein function and identification of new biomarkers and therapeutic targets for the diagnosis and treatment of human disease. These issues have engendered a new breed of comparative proteomic strategies, in which the complex array of proteins expressed by a given biological sample are parceled into manageable fractions prior to analysis.; Described here is a chemical proteomics method, "activity-based protein profiling" (ABPP), that was developed to compliment conventional genomic and proteomic methods that focus on measuring abundance rather than activity. ABPP employs chemical probes to covalently label active sites of enzyme superfamilies in a manner that provides a direct readout of changes in catalytic activity. By providing a covalent link between labeled proteins and a chemical tag, ABPP permits the consolidated detection, isolation, and identification of active enzymes directly from samples of high biological complexity.; This work represents the application of ABPP for the functional analysis of human cancer. First, we demonstrate the utility of ABPP in classifying phenotypically distinct classes of human cancer lines based on enzyme activity profiles of their secreted and membrane proteomes. These studies are then extended to the profiling of primary human tumors as well as an established mouse model of human cancer, demonstrating the dramatic functional differences that exist between cancer cells grown in culture or in the context of in vivo tumor growth, both of which serve as important, widely used, research models of human cancer. Also shown, is the use of ABPP to assign novel constituents to otherwise well characterized enzyme families. Finally, the development and application of activity-based probes directed at the metalloprotease family of enzymes is described.; Collectively, these studies establish ABPP as a powerful strategy for the functional analysis of large panels of cells/tissues, or to obtain in-depth information on a few samples, or even individual enzymes.
机译：考虑到蛋白质几乎介导了细胞，机体生理学和病理生理学的所有生化事件，因此开发直接测量细胞和组织蛋白质组中这些生物分子的水平和活性的通用方法的需求是显而易见的。基于活性分析蛋白质类别的能力将大大加速蛋白质功能的分配以及新的生物标志物和人类疾病的诊断和治疗靶标的鉴定。这些问题催生了一种新的比较蛋白质组学策略，其中在分析之前，将由给定的生物样品表达的复杂蛋白质阵列分解成可管理的级分。这里介绍的是一种化学蛋白质组学方法，即“基于活性的蛋白质谱分析”（ABPP），其开发目的是为了补充侧重于测量丰度而非活性的常规基因组和蛋白质组学方法。 ABPP使用化学探针以直接读出催化活性变化的方式共价标记酶超家族的活性位点。通过在标记的蛋白质和化学标签之间提供共价连接，ABPP可以直接从生物复杂性高的样品中直接检测，分离和鉴定活性酶。这项工作代表了ABPP在人类癌症功能分析中的应用。首先，我们证明了ABPP在根据其分泌蛋白和膜蛋白质组的酶活性谱对表型不同的人类癌症细胞系进行分类中的效用。然后，这些研究扩展到原发性人类肿瘤的概况分析以及已建立的人类癌症小鼠模型，证明了在培养物中或在体内肿瘤生长的情况下存在的癌细胞之间存在着巨大的功能差异，两者均起着作用。作为重要的，广泛使用的人类癌症研究模型。还显示了使用ABPP将新的成分分配给其他特征明​​确的酶家族。最后，描述了针对金属蛋白酶家族的基于活性的探针的开发和应用。总而言之，这些研究将ABPP确立为对大量细胞/组织进行功能分析，或获取有关少量样品甚至单个酶的深入信息的有力策略。

著录项

  • 作者

    Jessani, Nadim.;

  • 作者单位

    The Scripps Research Institute.;

  • 授予单位 The Scripps Research Institute.;
  • 学科 Biology Cell.; Biology Molecular.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 176 p.
  • 总页数 176
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;分子遗传学;
  • 关键词

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