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

描述（由申请人提供）：了解蛋白质功能需要了解蛋白质的定位和运输。 这在神经元中特别重要，因为它们具有复杂的形态和极化形状。 用同位素或荧光标记物标记蛋白质以追踪它们作为生物标志物已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、运输和信号传导可能会受到影响，需要进行严格的验证，以便对用生物标志物获得的数据具有信心。 目前，人们对量子点（QD）的使用有很大的热情和期望，因为它们具有明亮的荧光，差分光谱，极好的分辨率，并且它们是电子致密的，允许直接超微结构定位。 然而，QD标签的大小与小蛋白质相当，最近的证据表明QD与蛋白质的缀合可以改变标记蛋白质的功能和运输。 两个具有独特和互补专业知识的实验室之间的计划合作提供了探索和回答这些问题的机会。 我们建议从几个不同的蛋白质类与相应的“正常”蛋白质，最低限度地修改放射性碘化的八个量子点标记的蛋白质的行为进行比较。 我们的建议需要三种方法，是新颖的，在他们的组合：比较蛋白质贩运与“金标准”校准，检查在两个有利的体内模型系统，并在最高分辨率的评价-超微结构（电子显微镜）水平。 这项工作将产生一个逐步的方法学论文，将为QD标记的蛋白质作为生物标志物的用户提供如何验证其QD标记的蛋白质的指南。 我们还将确定最适合用作生物标志物的最佳缀合方案和QD大小。 虽然我们对神经元蛋白质运输和信号传导以及使用QD标记分子作为诊断和治疗工具的问题最感兴趣，但预期结果应该与广泛的组织类型和细胞生物学问题相关，这些问题超出了神经科学中生物标志物的使用。 公共卫生相关性：量子点标记的蛋白质在基础研究中有很大的前景，它们被提议作为诊断和治疗的生物医学工具。 在将其用作生物标志物之前，必须在体内模型系统中进行验证，以鉴定可能施加固有限制并损害最佳效用的运输、动力学、信号传导和受体结合的变化。

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.