Pocsfalvi Gabriella, Stanly Christopher, Fiume Immacolata, Vékey Károly
Mass Spectrometry and Proteomics, Institute of Biosciences and BioResources, National Research Council of Italy, Naples, Italy.
Mass Spectrometry and Proteomics, Institute of Biosciences and BioResources, National Research Council of Italy, Naples, Italy.
J Chromatogr A. 2016 Mar 25;1439:26-41. doi: 10.1016/j.chroma.2016.01.017. Epub 2016 Jan 11.
Extracellular vesicles (EVs), such as exosomes, microvesicles and apoptotic bodies are released by cells, both under physiological and pathological conditions. EVs can participate in a novel type of intercellular communication and deliver cargo of nucleic acids, proteins and lipids near or to distant host cells. EV research is proceeding at a fast pace; now they start to appear as promising therapeutic targets, diagnostic tools and drug delivery systems. Isolation and analysis of EVs are prerequisites for understanding their biological roles and for their clinical exploitation. In this process chromatography and mass spectrometry (MS)-based strategies are rapidly gaining importance; and are reviewed in the present communication. Isolation and purification of EVs is mostly performed by ultracentrifugation at present. Chromatography-based strategies are gaining ground, among which affinity and size exclusion chromatography (SEC) are particularly strong contenders. Their major advantages are the relative simplicity, robustness and throughput. Affinity chromatography has the added advantage of separating EV subtypes based on molecular recognition of EV surface motifs. SEC has the advantage that isolated EVs may retain their biological activity. EVs are typically isolated in small amounts, therefore high sensitivity is required for their analysis. Study of the molecular content of EVs (all compounds beside nucleic acids) is predominantly based on liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The chromatographic separation is mostly performed by reverse phase, nanoscale, ultra high performance LC technique. The MS analysis relying typically on nano-electrospray ionization MS/MS provides high sensitivity, selectivity and resolution, so that thousand(s) of proteins can be detected/identified/quantified in a EV sample. Beside protein identification, quantitation and characterization of protein post-translational modifications (PTMs), like glycosylation and phosphorylation are becoming feasible and increasingly important. Along with conventional LC-MS/MS, other chromatographic approaches hyphenated to MS are gaining importance for EV characterization. Hydrophilic interaction LC is used to characterize PTMs; LC-inductively coupled plasma/MS to identify metal containing molecules; while gas chromatography-MS to analyze some lipids and metabolites.
细胞外囊泡（EVs），如外泌体、微囊泡和凋亡小体，在生理和病理条件下均可由细胞释放。EVs能够参与一种新型的细胞间通讯，并将核酸、蛋白质和脂质等物质运输至附近或远处的宿主细胞。EV研究正在迅速发展；如今，它们开始展现出作为有前景的治疗靶点、诊断工具和药物递送系统的潜力。EVs的分离和分析是理解其生物学作用以及临床应用的先决条件。在此过程中，基于色谱法和质谱（MS）的策略正迅速变得重要起来；本文将对此进行综述。目前，EVs的分离和纯化大多通过超速离心法进行。基于色谱的策略正在逐渐普及，其中亲和色谱和尺寸排阻色谱（SEC）是特别有力的竞争者。它们的主要优点是相对简单、稳健且通量高。亲和色谱的额外优势在于能够基于对EV表面基序的分子识别来分离EV亚型。SEC的优势在于分离得到的EVs可能保留其生物活性。EVs通常分离量较少，因此其分析需要高灵敏度。对EVs分子内容物（除核酸外的所有化合物）的研究主要基于液相色谱串联质谱（LC-MS/MS）分析。色谱分离大多通过反相、纳米级、超高效液相色谱技术进行。通常依赖于纳升电喷雾电离MS/MS的质谱分析提供了高灵敏度、选择性和分辨率，从而能够在一个EV样品中检测/鉴定/定量数千种蛋白质。除了蛋白质鉴定、定量以及蛋白质翻译后修饰（PTM）（如糖基化和磷酸化）的表征外，这些研究也变得可行且日益重要。除了传统的LC-MS/MS，其他与质谱联用的色谱方法对于EV表征也越来越重要。亲水相互作用液相色谱用于表征PTM；液相色谱 - 电感耦合等离子体质谱用于鉴定含金属分子；而气相色谱 - 质谱用于分析一些脂质和代谢物。
