You Yang, Zhang Zhengrong, Cortes Samuel, Nguyen Son N, Vadakattu Prakruthi, Melvin Bridgette C, Jr Mann Sean D, Ray Nibedita Basu, Bregendahl Maria, McLean Pam J, Gonzalez-Perez Maria Paz, Ikezu Seiko, Ikezu Tsuneya
Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.
Department of Biomedical Education and Anatomy, The Ohio State University, Columbus, OH, USA.
Cell Commun Signal. 2025 May 30;23(1):253. doi: 10.1186/s12964-025-02263-3.
Extracellular vesicles (EVs) are critical mediators of intercellular communication by transferring proteins, lipid and nucleic acids between cells. EVs in biofluids, particularly blood, have gathered significant interest as potential biomarkers for disease diagnosis. However, isolating EVs from blood poses a challenge due to the high concentration of plasma proteins, which obscure the detection of low abundant EV-associated proteins. Here, we optimized a simplified and efficient method for isolating plasma-derived EVs by combining size exclusion chromatography (SEC) with flow-through chromatography using Capto Core 700 beads. A brief incubation of SEC-derived EV fractions with Capto Core beads (qEV + CC) enabled us to isolate intact, high-purity EVs with reduced soluble plasma protein contamination. As a comparison, MagReSyn-based method was not compatible with elution of intact EVs after the purification and showed significant contamination of soluble plasma proteins. Data-independent acquisition-based liquid chromatography-mass spectrometry of isolated plasma-EVs using the qEV + CC approach identified over 1,000 EV-associated proteins, including an increased presence of brain derived proteins and markers linked to neurodegenerative diseases, such as amyloid precursor protein and apolipoprotein E. These findings were further validated by super-resolution microscopy at a single EV resolution. Bioinformatic pathway and network analyses revealed enrichment of pathways involved in RNA processing, cell adhesion and synaptic function, highlighting the potential of EV molecules for broad disease biomarker discovery. Our findings present an optimized method for efficient purification of plasma-derived EVs, providing a valuable tool for advancing EV-based biomarker development.
细胞外囊泡（EVs）是细胞间通讯的关键介质，通过在细胞之间传递蛋白质、脂质和核酸来实现。生物流体中的EVs，尤其是血液中的EVs，作为疾病诊断的潜在生物标志物已引起了极大的关注。然而，由于血浆蛋白浓度高，从血液中分离EVs具有挑战性，这会掩盖低丰度的与EV相关的蛋白质的检测。在这里，我们通过将尺寸排阻色谱法（SEC）与使用Capto Core 700珠子的流通色谱法相结合，优化了一种简单有效的分离血浆来源的EVs的方法。将SEC衍生的EV级分与Capto Core珠子进行短暂孵育（qEV + CC），使我们能够分离出完整、高纯度的EVs，同时减少可溶性血浆蛋白的污染。作为比较，基于MagReSyn的方法在纯化后与完整EVs的洗脱不兼容，并且显示出可溶性血浆蛋白的显著污染。使用qEV + CC方法对分离的血浆EVs进行基于数据非依赖采集的液相色谱-质谱分析，鉴定出超过1000种与EV相关的蛋白质，包括脑源性蛋白质和与神经退行性疾病相关的标志物（如淀粉样前体蛋白和载脂蛋白E）的含量增加。这些发现通过单EV分辨率的超分辨率显微镜进一步得到验证。生物信息学通路和网络分析揭示了参与RNA加工、细胞粘附和突触功能的通路的富集，突出了EV分子在广泛疾病生物标志物发现中的潜力。我们的发现提出了一种优化的方法，用于高效纯化血浆来源的EVs，为推进基于EV的生物标志物开发提供了有价值的工具。
