RII Track-4:NSF: ExoPRIME Technology and Mass Spectroscopy Proteomic Analysis of Immunocaptured Exosomes for Liquid Biopsy

细胞外囊泡是由细胞分泌的含有蛋白质和核酸的纳米颗粒，是一种很有前途的液体活检工具。循环EV的蛋白质组学分析是用于病理状况的精确和非侵入性早期诊断的新兴方法。选择性分离表达相同表面抗体的EV亚群对于精确鉴定疾病特异性蛋白质组学生物标志物至关重要。这项拟议的研究将使技术的特异性评估能够直接纯化和蛋白质组学分析表达相同表面标志物的EV亚群，因此来源于患病组织。这项工作的结果也将提高我们对癌症起源EV的生物标志物特异性特征的理解，以通过液体活检方法进行早期诊断。该奖学金计划将培训PI和路易斯安那理工大学的一名研究生，包括质谱样品处理的技术方面以及高通量蛋白质组学数据分析的计算方法。这将使PI能够现代化并提高路易斯安那理工大学蛋白质组学课程的严谨性。这个研究基础设施改善轨道-4 EPSCoR研究员（RII轨道-4）项目将提供奖学金的助理教授和研究生在路易斯安那理工大学。这项工作将与休斯顿大学的研究人员合作进行。使用疾病特异性表面标志物从复杂生物流体中选择性免疫纯化EV提供了用于早期疾病诊断的非侵入性方法。在该项目中，PI和一名研究生将访问休斯顿大学的质谱设施，利用布鲁克的timsTOF Pro质谱系统对星形胶质细胞和肿瘤标志物特异性（EpCAM，EGFRvIII和HSP-27）胶质母细胞瘤分泌的EV进行自下而上的质谱蛋白质组学分析。PI已经证明了免疫捕获EV工具的可行性。ExoPRIME方法的核心是抗体功能化不锈钢显微针（3 mm ×130µm），用于纯化表达特定表面标记的EV。这项研究的主要目的是确定胶质母细胞瘤特异性蛋白质组学生物标志物，并评估ExoPRIME工具在护理点进行临床诊断的可行性。该项目将实现以下目标：1）评估使用商业免疫捕获方法纯化的胶质母细胞瘤和健康星形胶质细胞衍生EV的蛋白质组学谱; 2）使用ExoPRIME技术纯化的胶质母细胞瘤衍生EpCAM、EGFRvIII和HSP-27特异性EV的蛋白质组学分析。在此奖学金期间获得的专业知识也将使PI能够培训和教育路易斯安那理工大学的学生在尖端的蛋白质组学分析技术。这项研究有可能导致对肿瘤特异性EV的蛋白质组学特征的全面了解，从而为生物标志物发现和早期疾病诊断的技术开发打开大门。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Extracellular vesicles (EVs) are nanosized particles secreted by the cells that contain protein and nucleic acid and are a promising tool for liquid biopsy. Proteomics analysis of circulating EVs is an emerging method for precise and non-invasive early diagnosis of pathological conditions. Selective isolation of EV subpopulations that express the same surface antibody is critical for the precise identification of disease-specific proteomics biomarkers. This proposed research will enable the specificity assessment of technology for direct purification and proteomic analysis of subpopulations of EVs that express the same surface marker, and therefore originate from diseased tissue. The results from this work will also improve our understanding of biomarkers-specific signatures of cancer-originating EVs for early diagnostics via a liquid biopsy approach. The fellowship program will train the PI and a graduate student from Louisiana Tech University in both technical aspects of sample processing for mass spectrometry as well as computational approaches for high throughput proteomics data analysis. This will enable the PI to modernize and increase the rigor of the proteomics courses at Louisiana Tech University. This Research Infrastructure Improvement Track-4 EPSCoR Research Fellows (RII Track-4) project would provide a fellowship to an Assistant Professor and a graduate student at Louisiana Tech University. This work would be conducted in collaboration with researchers at the University of Houston. Selective immunopurification of EVs from complex biological fluids using disease-specific surface markers provides a non-invasive method for early disease diagnosis. In this project, the PI and one graduate student will visit the Mass Spectrometry Facility at the University of Houston to perform bottom-up mass spectrometry proteomic analysis of astrocytes and tumor marker specific (EpCAM, EGFRvIII, and HSP-27) glioblastoma-secreted EVs utilizing Bruker's timsTOF Pro mass spectrometry system. The PI has demonstrated the feasibility of a tool for immunocapture of EVs. At the core of the ExoPRIME approach is an antibody-functionalized stainless steel microscopic pin (3mm×130µm) for the purification of EVs expressing a specific surface marker. The main objectives of this research study are to identify glioblastoma-specific proteomics biomarkers and assess the feasibility of the ExoPRIME tool for clinical diagnostics at the point of care. This project will achieve the following objectives:1) assess the proteomics profile of glioblastoma and healthy astrocytes-derived EVs purified using a commercial immunocapture approach; and 2) perform proteomics analysis of glioblastoma-derived EpCAM, EGFRvIII, and HSP-27 specific EVs purified using the ExoPRIME technology. Expertise gained during this fellowship will also enable the PI to train and educate students at Louisiana Tech University in cutting-edge proteomics analysis techniques. This research has potential to lead to a comprehensive understanding of the proteomics profile of tumor-specific EVs which opens the door to technology development for biomarker discovery and early disease diagnostics.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.