Quantitative imaging of the dystrophin-glycoprotein complex

项目摘要 许多令人兴奋的治疗干预杜氏肌营养不良症正在出现，但挑战， 定量功能性肌营养不良蛋白使药物审批复杂化并延迟了临床转化的步伐。 为了克服这一障碍，我们正在开发一种多重成像-质谱（I-MS）方法， 同时定量和定位单个肌肉切片中的肌营养不良蛋白和DGC的其他成员。 我们的初步数据显示成功的定量和定位的肌营养不良蛋白和肌球蛋白在小鼠 肌肉切片支持我们提出的方法的可行性。这种方法改进了定量 蛋白质印迹，同时提供了肌营养不良蛋白和其他DGC蛋白的定位， 以前通过单独的免疫组织化学进行。 具体目标1：单克隆抗体的稀土元素标记抗体的开发和验证 肌营养不良蛋白和其他DGC组分的成像-质谱。32种一抗，DNA 嵌入剂或凝集素将用特异性RE标记。将对每个RE标记的抗体进行验证 通过比较Western印迹、免疫荧光和I-MS，使用野生型、杂合mdx， 纯合mdx小鼠和正常人肌肉样品。这一目标将：（1）建立一个强大的管道， 用于I-MS的抗体标记、质量控制和验证，2）对这种新方法进行基准测试， 常规方法，以及3）为目标2中的多重I-MS提供所需的坚实基础。 具体目标2：肌营养不良蛋白和其他DGC组分的多重成像-质谱分析。我们 将从目标1中组装一组经验证的抗体，并使用它们同时测量 肌营养不良蛋白和其它DGC组分。我们选择了不同的组织 抗肌萎缩蛋白和DGC组分水平，包括：1）转基因小鼠模型， 2）突变型肌营养不良蛋白基因的患者携带者和3）Becker氏肌营养不良症患者 营养不良该目标将：1）优化和验证目标1中试剂的多重化，2）测试灵敏度 该方法在临床相关样本上的有效性和3）证明临床可翻译性。 我们的新应用成像质谱将有助于绝对定量和肌膜 在非常小的肌肉样品中定位功能性肌营养不良蛋白和多达32个DGC成员。临床 DMD的研究将受益于准确定量和成像功能性肌营养不良蛋白的能力， DGC将充分利用宝贵的患者资源，提供更好的药物信息 有效性和安全性，并加快临床转化的步伐。

PROJECT SUMMARY Many exciting therapeutic interventions for Duchenne muscular dystrophy are emerging, but the challenges of quantitating functional dystrophin are complicating drug approvals and delaying the pace of clinical translation. To overcome this obstacle, we are developing a multiplexed imaging-mass spectrometry (I-MS) method to simultaneously quantitate and localize dystrophin and other members of the DGC in single muscle sections. Our preliminary data showing successful quantitation and localization of dystrophin and myosin in mouse muscle sections supports the feasibility of our proposed approach. This approach improves on the quantitation of Western blots and simultaneously provides the localization of dystrophin and other DGC proteins that was previously done by separate immunohistochemistry. Specific Aim 1: Development and validation of rare earth element labeled antibodies for simplex imaging-mass spectrometry of dystrophin and other DGC components. 32 primary antibodies, DNA intercalators or lectins will be labeled with specific REEs. Each REE-labeled antibody will be validated individually by comparing Western blot, immunofluorescence and I-MS using wild-type, heterozygous mdx, homozygous mdx mouse and normal human muscle samples. This aim will: 1) develop a robust pipeline for antibody labeling, quality control and validation for use in I-MS, 2) benchmark this new approach to conventional approaches, and 3) provide the solid foundation required for multiplex I-MS in Aim 2. Specific Aim 2: Multiplex imaging-mass spectrometry of dystrophin and other DGC components. We will assemble a panel of the validated antibodies from Aim 1 and use them to simultaneously measure dystrophin and other DGC components in single muscle tissue sections. We have selected tissues of varying dystrophin and DGC component levels including: 1) transgenic mouse models engineered for increasing levels of human sarcospan, 2) patient carriers of a mutant dystrophin gene and 3) patients with Becker's muscular dystrophy. This aim will: 1) optimize and validate the multiplexing of reagents from Aim 1, 2) test the sensitivity of this approach on clinically relevant samples and 3) demonstrate clinical translatability. Our novel application of imaging-mass spectrometry will facilitate the absolute quantitation and sarcolemmal localization of functional dystrophin and up to 32 DGC members in very small muscle samples. Clinical research in DMD will benefit from the ability to accurately quantitate and image functional dystrophin and the DGC, which will make the best use of valuable patient resources, provide improved information on drug efficacy and safety, and speed the pace of clinical translation.