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。由于神经元的复杂形态和极化形状，这在神经元中尤为重要。用同位素或荧光标记标记蛋白质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损害，为了对生物标记物获得的数据有信心，需要严格的验证。目前，人们对量子点（QDs）的使用有着极大的热情和期望，因为它们具有明亮的荧光，微分光谱，极好的分辨率，并且它们是电子密集的，允许直接的超微结构定位。然而，QD标签的大小与小蛋白质相当，最近的证据表明，与蛋白质结合的QD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8种量子点标记蛋白质与相应的经放射性碘化最小修饰的“正常”蛋白质的行为。我们的建议涉及三种新颖的方法组合：蛋白质运输与“金标准”校准的比较，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。这项工作将导致一步一步的方法学论文，将为qd标记的蛋白质作为生物标志物的用户提供指导，如何验证其qd标记的蛋白质。我们还将确定最适合用作生物标志物的最佳偶联方案和QD大小。虽然我们最感兴趣的是神经元蛋白运输和信号传导的问题，以及使用量子点标记的分子作为诊断和治疗工具，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，而不仅仅是生物标志物在神经科学中的应用。

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.