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

验证使用量子点标记蛋白作为体内生物标志物

基本信息

批准号：
8130807
负责人：
CHRISTOPHER S VON BARTHELD
金额：
$ 17.65万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8130807
关键词：
Acceleration Acute Amyloid beta-Protein Precursor Axon Axonal Transport Basic Science Behavior Biological Biological Assay Biological Markers Biological Models Calibration Cells Chemicals Chick Embryo Collaborations Complex Data Defect Diagnosis Diagnostic Electrons Embryo Enzyme-Linked Immunosorbent Assay Evaluation Fluorescence Gel Gold Growth Factor Guidelines Health Histocompatibility Testing Hypoglossal nerve structure Interruption Investigation Iodination reaction Isotopes Kinetics Knowledge Label Lectin Location Lysosomes Microscopic Morphology Motor Neurons Multivesicular Body Nanotechnology Neurons Neurosciences Organelles Paper Pathway interactions Presynaptic Terminals Prions Process Proteins Protocols documentation Quantum Dots Radio Radiolabeled Rattus Research Personnel Resolution Scheme Science Shapes Signal Transduction Solutions Staging Testing Therapeutic Time Toxic effect Validation Visual system structure Work behavioral impairment chemical synthesis design deviant expectation in vivo in vivo Model indexing interest light scattering neuronal cell body neurotrophic factor novel strategies postnatal protein complex protein degradation protein function protein transport public health relevance radiotracer receptor binding research study tetanus toxin fragment C tool trafficking uptake wasting

项目摘要

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.

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。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。用同位素或荧光标签标记蛋白质以追踪它们，因为生物标志物已成为生物医学科学中广泛使用的工具。但是，当蛋白质通过TAG修饰时，正常蛋白质相互作用，运输和信号传导可能会受到损害，并且需要严格验证才能对使用生物标志物获得的数据有信心。当前，对于使用量子点（QD）的热情和期望，因为它们具有明亮的荧光，差异光谱，出色的分辨率，并且它们是电子密度，从而可以直接进行超微结构定位。但是，QD标签的大小与小蛋白质相当，最近的证据表明，QD结合蛋白可以改变标记蛋白的功能和运输。具有独特和互补专业知识的两个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。我们建议将来自几种不同蛋白质类别的八种QD标记蛋白的行为与相应的“正常”蛋白质的行为进行比较，并通过射线固化进行了最小化。我们的建议需要三种新颖的方法：将蛋白质运输与“黄金标准”校准进行比较，两种有利的体内模型系统的检查以及最高分辨率的评估 - 超微结构（电子显微镜）水平。这项工作将导致逐步的方法论论文，该论文将为QD标记蛋白的用户提供指南，作为生物标志物如何验证其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共轭方案和QD大小。虽然我们对这个问题最感兴趣的是神经元蛋白运输和信号传导，以及使用QD标记的分子作为诊断和治疗工具，但预期结果应与广泛的组织类型和细胞生物学问题有关神经科学。公共卫生相关性：量子点标记的蛋白质在基础研究中具有巨大的希望，并被认为是诊断和治疗生物医学工具。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，以确定可能施加固有局限性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。