Adav Sunil S, Sze Siu Kwan
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.
Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.
Methods Mol Biol. 2017;1619:193-201. doi: 10.1007/978-1-4939-7057-5_15.
To detect disease at an early stage and to develop effective disease treatment therapies, reliable biomarkers of diagnosis, disease progression, and its status remain a research priority. A majority of disease pathologies are primarily associated with different subsets of cells of different tissues, discrete compartments, and areas. These subsets of cells release glycoproteins and specific extracellular vesicles (EVs) including microvesicles and exosomes that carry bioactive cargoes of proteins, nucleic acids, and metabolites. Body fluids like blood plasma are considered as a golden source of disease biomarkers since it contains glycoprotein and EVs released by almost all cell types. The contents of glycoproteome and EV cargo change with cell status, and they act as mirror of cell's intracellular events and status; hence, EVs and glycoproteins are promising disease biomarkers. However, their abundance in blood plasma remains low posing a serious technical problem in their identification and quantification. Until recently, technical advances and exhaustive research devised a technique for either enrichment of plasma glycoprotein or EVs, but no methodologies exist that can enrich and identify both plasma glycoprotein and EVs. To overcome this technical challenge, a method that can eliminate high-abundance entities without depleting disease-modifying molecules is required. Therefore, here we describe the detailed protocol of simultaneous enrichment of glycoproteins and EVs from blood plasma by prolonged ultracentrifugation coupled to electrostatic repulsion-hydrophilic interaction chromatography (PUC-ERLIC) and their identification and quantification by mass spectrometry-based proteomic technique.
为了在疾病早期进行检测并开发有效的疾病治疗方法，可靠的诊断、疾病进展及其状态的生物标志物仍然是研究的重点。大多数疾病病理主要与不同组织、离散区室和区域的不同细胞亚群相关。这些细胞亚群释放糖蛋白和特定的细胞外囊泡（EVs），包括携带蛋白质、核酸和代谢物生物活性货物的微囊泡和外泌体。像血浆这样的体液被认为是疾病生物标志物的黄金来源，因为它含有几乎所有细胞类型释放的糖蛋白和EVs。糖蛋白组和EV货物的内容随细胞状态而变化，它们就像细胞内事件和状态的镜子；因此，EVs和糖蛋白是很有前景的疾病生物标志物。然而，它们在血浆中的丰度仍然很低，这在它们的鉴定和定量方面带来了严重的技术问题。直到最近，技术进步和详尽的研究设计了一种用于富集血浆糖蛋白或EVs的技术，但不存在能够同时富集和鉴定血浆糖蛋白和EVs的方法。为了克服这一技术挑战，需要一种能够消除高丰度实体而不消耗疾病修饰分子的方法。因此，在这里我们描述了通过长时间超速离心结合静电排斥-亲水相互作用色谱法（PUC-ERLIC）从血浆中同时富集糖蛋白和EVs的详细方案，以及通过基于质谱的蛋白质组学技术对它们进行鉴定和定量的方法。
