Taleb Raghda Saad Zaghloul, Moez Pacint, Younan Doreen, Eisenacher Martin, Tenbusch Matthias, Sitek Barbara, Bracht Thilo
Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
Clinical and Chemical Pathology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt.
Methods Mol Biol. 2019;1959:51-64. doi: 10.1007/978-1-4939-9164-8_4.
Cells shed into the extracellular space a population of membranous vesicles of plasma membrane origin called microparticles (MP). Given the fact that MP are abundantly present in body fluids including plasma, rich in cell-type or disease-specific proteins and formed in conditions of stress and injury, they have been extensively investigated as biomarkers in various diseases. With the advancement in the mass spectrometry-based proteome analysis, the knowledge of the protein composition of plasma MP (PMP) has been intensively expanded, which aids the discovery of novel diagnostic target proteins. However, the lack of standardized and accurate protocols for PMP isolation limits the implementation of PMP as biomarkers in clinical settings. Here, we describe in detail a robust protocol for PMP isolation from human blood plasma via ultracentrifugation followed by label-free quantitative proteome analysis of PMP.
细胞向细胞外空间释放一群源自质膜的膜性囊泡，称为微粒（MP）。鉴于MP大量存在于包括血浆在内的体液中，富含细胞类型或疾病特异性蛋白质，且在应激和损伤条件下形成，它们已被广泛研究作为各种疾病的生物标志物。随着基于质谱的蛋白质组分析技术的进步，血浆MP（PMP）蛋白质组成的知识得到了深入扩展，这有助于发现新的诊断靶蛋白。然而，缺乏标准化和准确的PMP分离方案限制了PMP在临床环境中作为生物标志物的应用。在此，我们详细描述了一种通过超速离心从人血浆中分离PMP的可靠方案，随后对PMP进行无标记定量蛋白质组分析。
