Name: Proteome/Phosphoproteome Analysis of the Extracellular Vesicle (EV) Samples Using LC-MS/MS
Uploaded: 2023-10-27T13:10:00
Description: Watch this Scientific Journal Video about Proteome/Phosphoproteome Analysis of the Extracellular Vesicle EV Samples Using LC-MS/MS at JoVE.com

proteome/phosphoproteome analysis of the extracellular vesicle (ev) samples using lc-ms/ms

增强 EV 分离以进行精确的生物流体分析

Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles secreted by all types of cells and are present in biofluids such as blood, urine, saliva, etc.1,2,3,4. EVs carry a cargo of diverse bioactive molecules which reflect the physiological and pathological state of their host cells and, therefore function as crucial factors in disease progression4,5,6. Moreover, extensive studies have established that EV-based disease markers can be identified prior to the onset of symptoms or the physiological detection of tumors5,6,7.
Phosphorylation acts as a key mechanism in cellular signaling and regulation. Therefore, phosphoproteins provide a valuable source for biomarker discovery as aberrant phosphorylation events are associated with dysregulated cellular signaling pathways and metastatic disease development such as cancer8,9,10. Although profiling phosphorylation dynamics allows for the identification of disease-specific phosphoprotein signatures as potential biomarkers, the low abundance and dynamic nature of phosphoproteins pose major challenges in developing phosphoproteins as biomarkers11,12. Notably, the low-abundant phosphoproteins encapsulated within EVs are protected from external enzymatic digestion in the extracellular environment8. Consequently, EVs and EV-derived phosphoproteins offer an ideal source for biomarker discovery in the early-stage detection of cancer and other diseases.
Although analysis of protein phosphorylation in EVs offers a valuable resource for understanding cancer signaling and early-stage disease diagnosis, the lack of efficient EV isolation methods presents a major barrier. EV isolation is commonly achieved through differential ultracentrifugation (DUC)13. However, this method is time-consuming and is not suitable for clinical implications due to low throughput and poor reproducibility13,14. Alternative EV isolation approaches, such as polymer-induced precipitation15, are limited by low specificity due to co-precipitation of non-EV proteins. Affinity-based approaches, including antibody-based affinity capture16 and affinity filtration17, offer enhanced specificity but are restricted to a relatively low recovery rate due to small volume.
To address the issues in exploring phosphoprotein dynamics in EVs, our group has developed extracellular vesicles total recovery and purification (EVtrap) technique based on chemical affinity to capture EVs onto functionalized magnetic beads18. Previous results have demonstrated that this magnetic bead-based EV isolation method is highly effective in isolating EVs from a wide range of biofluid samples and is able to achieve much higher EV yield while minimizing contamination compared to DUC and other existing isolation methods18,19. We have successfully utilized EVtrap and a titanium-based phosphopeptide enrichment method developed by our group20 to profile the phosphoproteome of EVs derived from diverse biofluids and to detect potential phosphoprotein biomarkers for various diseases19,21,22.
Here, we present a protocol based on EVtrap for the isolation of circulating EVs. The protocol focuses on the urinary EVs. We also demonstrate the characterization of isolated EVs using western blotting. We then detail the sample preparation and mass spectrometry (MS) acquisition for both proteomics and phosphoproteomics analyses. This protocol provides an efficient and reproducible workflow for profiling the urinary EV proteome and phosphoproteome, which will facilitate further studies on EVs and their clinical applications23.

作者聚焦：在疾病生物标志物发现中推进 EVtrap 用于高通量蛋白质组学 

基于化学亲和力的生物流体细胞外囊泡分离用于蛋白质组学和磷酸化蛋白质组学分析

We are working on extracellular vesicle-based biomarker discovery. One important step is to isolate them efficiently and selectively. EVtrap, which is based on magnetic bead-based separation, can really help us by providing high throughput, easy to use, in particular, easy for clinical applications.We have been using this protocol for discovery in proteins and the phosphoprotein biomarkers for different diseases such as breast cancer, kidney cancers, and Parkinson's Disease. This biomarker is derived from biofluid. EVs can potentially be used for liquid biopsies, which can further improve clinical diagnosis and the treatment.Currently, the most commonly used approach for extracellular vesicle isolation are ultracentrifugation and precipitation, but those methods are limited to co-isolation of contaminants and also low efficiency. So our EVtrap method will provide a more effective way for isolating EVs, which will allow for a more reproducible and sensitive method and also will benefit downstream analysis. We are working on two things.First, we are continue developing the methods and protocols for EV isolation and a downstream analysis, in particular, a mass backup-based analysis. Second one is we are exploring different application in clinical field because of the advantage by the EVtrap.

Watch this Scientific Journal Video about Proteome/Phosphoproteome Analysis of the Extracellular Vesicle EV Samples Using LC-MS/MS at JoVE.com

Proteome/Phosphoproteome Analysis of the Extracellular Vesicle (EV) Samples Using LC-MS/MS  

使用LC-MS/MS对细胞外囊泡（EV）样品进行蛋白质组/磷酸化蛋白质组分析

首先，使用 EVtrap 方法制备从尿液样本中分离的细胞外囊泡的蛋白质组和磷酸化蛋白质组样品。通过液相色谱系统将样品注入捕获的离子淌度飞行时间质谱仪中。将肽分离到15厘米的C18柱中。对于蛋白质组学分析，使用dia-PASEF方法获取数据，每个斜坡的质量范围为300至1200质荷比，离子迁移常数为0.6至1.50 1 1/Knot。对于磷酸化蛋白质组学分析，使用dia-PASEF采集方法采集数据。将每个斜坡的质量范围设置为 400 至 1550 质荷比，离子迁移常数为 0.6 至 1.50 1/Knot。将原始文件加载到蛋白质组学软件中。在智人数据库中设置搜索参数。对于“消化类型”，选择“特异性”，然后在“酶”下选择“胰蛋白酶P”。将肽长度定义为最小 7 和最大 52，并允许两次漏裂。然后将“最大变量修改数”设置为 5。固定修饰应为胱氨酸位点的脲甲基，而可变修饰应包括乙酰蛋白 N 端、蛋氨酸氧化以及丝氨酸、苏氨酸和酪氨酸的磷酸化。最后，将肽谱匹配、肽和蛋白质组的错误发现率设置为 0.01。在LCMS和MS蛋白质组学分析中，每个样品的2%揭示了来自大约2,200种蛋白质的11,000多种独特肽。值得注意的是，与 ExoCarta 数据库相比，在所有三个重复中都一致地发现了 72% 的独特蛋白质，并鉴定了 90 个顶级 EV 标记和蛋白质。定量精密度为5.7%的中低变异系数，表明具有高重现性和可靠性。对于磷酸化蛋白质组学，每个肽样品的 98% 产生了 800 个独特的磷酸肽和 350 个磷蛋白。富集得到72%的磷酸丝氨酸、22%的磷酸苏氨酸和6%的磷酸酪氨酸肽。在所有重复中鉴定出42%的磷酸肽，观察到21.8%的中等变异系数，表明可接受的定量重现性。

Research

JoVE Journal

Biochemistry

Proteome/Phosphoproteome Analysis of the Extracellular Vesicle (EV) Samples Using LC-MS/MS

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

科研

生物化学

Extracellular Vesicle (EV) Isolation from Human Urine Samples Using the EVtrap Approach

使用 EVtrap 方法从人尿液样本中分离细胞外囊泡 （EV）

Preparation of Dried Extracellular Vesicle (EV) Sample for Proteomics and Phosphoproteomics Analysis

用于蛋白质组学和磷酸化蛋白质组学分析的干燥细胞外囊泡 （EV） 样品的制备