Validation of the use of quantum dot-labeled proteins as biomarkers in vivo

描述(由申请人提供)：了解蛋白质的功能需要了解蛋白质的定位和运输。由于其复杂的形态和极化的形状，这在神经元中尤其重要。用同位素或荧光标记标记蛋白质作为生物标记物已经成为生物医学科学中广泛使用的工具。然而，当蛋白质被标签修饰时，正常的蛋白质相互作用、运输和信号传递可能会受到影响，需要严格的验证才能对通过生物标记物获得的数据有信心。目前，人们对量子点的使用抱有极大的热情和期待，因为它们具有明亮的荧光、差示光谱、极高的分辨率，并且它们是电子致密的，允许直接的超微结构定位。然而，QD标签在大小上与小蛋白相当，最近的证据表明，QD与蛋白质的结合可以改变标记蛋白质的功能和运输。两个拥有独特和互补专业知识的实验室之间计划中的协作为探索和回答这些问题提供了机会。我们建议比较来自几种不同蛋白质类别的八种QD标记的蛋白质与相应的“正常”蛋白质的行为，这些蛋白质通过放射性碘标记进行最小程度的修饰。我们的建议结合了三种新颖的方法：比较蛋白质运输与“金标准”校准，在两个有利的体内模型系统中进行检查，以及在最高分辨率-超微结构(电子显微镜)水平上进行评估。这项工作将产生一份循序渐进的方法学论文，为作为生物标记物的QD标记蛋白质的使用者提供如何验证其QD标记蛋白质的指南。我们还将确定最适合用作生物标志物的最佳连接方案和量子点大小。虽然我们最感兴趣的问题是神经元蛋白质的运输和信号传递，以及QD标记分子作为诊断和治疗工具的使用，但预期的结果应该与广泛的组织类型和细胞生物学问题相关，超越了神经科学中生物标记物的使用。 公共卫生相关性：量子点标记的蛋白质在基础研究中前景广阔，它们被建议作为诊断和治疗生物医学工具。在将它们用作生物标记物之前，必须在体内模型系统中进行验证，以确定可能施加固有限制并影响最佳效用的转运、动力学、信号和受体结合的变化。

DESCRIPTION (provided by applicant): Understanding protein function requires knowledge about a protein's localization and trafficking. This is particularly important in neurons due to their complex morphology and polarized shape. Tagging proteins with either isotopes or fluorescent tags to trace them as biomarkers has become a widely used tool in biomedical science. However, when proteins are modified by a tag, normal protein interactions, trafficking and signaling may be compromised, and rigorous validation is required in order to have confidence in the data obtained with the biomarker. Currently, there is great enthusiasm and expectations for the use of quantum dots (QDs), because they have bright fluorescence, differential spectra, superb resolution, and they are electron dense, allowing for direct ultrastructural localization. However, QD tags are comparable in size to small proteins, and recent evidence indicates that QD conjugation to proteins can alter the function and trafficking of tagged proteins. The planned collaboration between two labs with unique and complementary expertise provides the opportunity to explore and answer these questions. We propose to compare the behavior of eight QD-tagged proteins from several different protein classes with the corresponding "normal" protein, minimally modified by radio-iodination. Our proposal entails three approaches that are novel in their combination: comparison of protein trafficking with a "gold standard" calibration, examination in two advantageous in-vivo model systems, and evaluation at highest resolution - the ultrastructural (electron microscopic) level. This work will result in a step-by-step methodological paper that will provide guidelines for users of QD-tagged proteins as biomarkers how to validate their QD-tagged proteins. We will also determine optimal conjugation schemes and QD sizes that are most suitable for use as biomarkers. While we are most interested in this question for neuronal protein trafficking and signaling, and use of QD-labeled molecules as diagnostic and therapeutic tools, the expected results should be relevant for a wide range of tissue types and cell biological questions extending beyond biomarker use in neuroscience. PUBLIC HEALTH RELEVANCE: Quantum dot-labeled proteins have great promise in basic research and they are proposed as diagnostic as well as therapeutic biomedical tools. Prior to their use as biomarkers, validation has to occur in in-vivo model systems, to identify changes in trafficking, kinetics, signaling and receptor binding that may impose inherent limitations and compromise optimal utility.