从骨髓来源的巨噬细胞中鉴定小细胞外囊泡的肽
Summary
该协议描述了通过差异超速离心从巨噬细胞中分离小细胞外囊泡并提取肽圆顶以进行质谱鉴定的程序。
Abstract
小细胞外囊泡 （sEV） 通常由多泡体 （MVB） 的胞吐作用分泌。这些直径为<200nm的纳米囊泡存在于各种体液中。这些 sEV 通过其货物（如蛋白质、DNA、RNA 和代谢物）调节各种生物过程，例如基因转录和翻译、细胞增殖和存活、免疫和炎症。目前，已经开发了各种用于sEV分离的技术。其中，基于超速离心的方法被认为是金标准，广泛用于sEV分离。肽是天然生物大分子，长度少于50个氨基酸。这些肽参与具有生物活性的各种生物过程，如激素、神经递质和细胞生长因子。肽球用于通过液相色谱-串联质谱（LC-MS/MS）系统分析特定生物样品中的内源性肽。在这里，我们介绍了一种通过差示超速离心分离sEV的方案，并提取了肽球用于LC-MS / MS鉴定。该方法从骨髓来源的巨噬细胞中鉴定了数百种sEVs衍生的肽。
Introduction
直径小于 200 nm 的小细胞外囊泡 （sEV） 存在于几乎所有类型的体液中，并由各种细胞分泌，包括尿液、汗液、泪液、脑脊液和羊水1.最初，sEV被认为是处理细胞废物的容器，这导致随后十年的研究很少2。最近，越来越多的证据表明，sEV含有特定的蛋白质、脂质、核酸和其他代谢物。这些分子被转运到靶细胞3，有助于细胞间通讯，通过这些细胞参与各种生物过程，如组织修复、血管生成、免疫4和炎症5,6、肿瘤发育和转移7,8,9等。
为了促进sEV的研究，必须从复杂样品中分离sEV。根据sEV的物理和化学性质，如密度、粒径和表面标记蛋白，已经开发了不同的sEV分离方法。这些技术包括基于超速离心的方法、基于粒径的方法、基于免疫亲和捕获的方法、基于sEVs沉淀的方法和基于微流体的方法10,11,12。在这些技术中，基于超速离心的方法被广泛认为是sEV分离的金标准，也是最常用的技术13。
越来越多的证据表明，在各种生物体的肽层中存在大量未被发现的生物活性肽。这些肽通过调节生长、发育、应激反应14,15和信号转导16，显著促进许多生理过程。sEVs肽球的目的是揭示这些sEV携带的肽，并为它们的生物学功能提供线索。在这里，我们提出了通过差分超速离心分离sEV的方案，然后从这些sEV中提取肽以进一步分析其肽。
Protocol
1. 小细胞外囊泡的分离 注意：在4°C下执行步骤1.1-1.11中的所有离心。 制备不含sEVs的胎牛血清（FBS）：通过超速离心机（见材料表）在4°C下以110,000 ×g离心FBS过夜以除去内源性sEV。收集上清液，用0.2μm超滤膜过滤灭菌，并在-20°C下储存。 在150mm培养皿上平板约3 x 107 永生化骨髓来源巨噬细胞（iBMDM），并加入20 mL DMEM培养?…
Representative Results
对于通过差分超速离心分离的sEV（图1），我们根据国际细胞外囊泡学会（ISEV）17评估了它们的形态，粒度分布和蛋白质标记。 首先，通过透射电镜观察sEV的形态，显示出典型的杯状结构（图2A）。NTA显示，分离的sEV主要集中在136 nm处（图2B），这与报告的尺寸（30-150 nm）一致</s…
Discussion
在研究sEV的功能时，必须从复杂的生物样品中获得高纯度的sEV，以避免任何潜在的污染。已经开发了多种sEV分离方法13，在这些方法中，基于差示超速离心的方法显示出相对较高的sEV纯度。本研究收集200 mL细胞上清液6 h，差示超速离心获得约200-300 μgsEV。但是，应该注意的是，在超速离心过程中，sEVs沉淀可能不可见（步骤1.8）。因此，建议尽可能多地移液管底部。这一步至关重?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了中国自然科学基金（3157270）的资助。我们感谢邵峰博士（中国国家生命科学研究所）提供iBMDM。
Materials
| BCA Protein Assay Kit | Beyotime Technology | P0012 | |
| CD9 | Beyotime Technology | AF1192 | |
| Centrifugal filter tube | Millipore | UFC5010BK | |
| Centrifuge bottles polypropylene | Beckman Coulter | 357003 | High-speed centrifuge |
| Chemiluminescent substrate | Thermo Fisher Scientific | 34580 | |
| Dithiothreitol | Solarbio | D8220 | 100 g |
| DMEM culture medium | Cell World | N?A | |
| GRP94 | Cell Signaling Technology | 20292 | |
| High-speed centrifuge | Beckman Coulter | Avanti JXN-26 | Centrifuge rotor (JA-25.50) |
| Immortalized bone marrow-derived macrophages (iBMDM) | National Institute of Biological Sciences, China | Provided by Dr. Feng Shao (National Institute of Biological Sciences, China) | |
| Iodoacetamide | Sigma | l1149 | 5 g |
| Microfuge tube polypropylene | Beckman Coulter | 357448 | 1.5 mL, Tabletop ultracentrifuge |
| nano-high-performance LC system | Thermo Fisher Scientific | EASY-nLC 1000 | |
| Nanoparticle tracking analysis | Malvern Panalytical | NanoSight LM10 | NanoSight NTA3.4 |
| Orbitrap Q Exactive HF-X mass spectrometer | Thermo Fisher Scientific | N/A | |
| Phosphate-buffered saline | Solarbio | P1020 | |
| Polyallomer centrifuge tubes | Beckman Coulter | 326823 | Ultracentrifuge |
| Protease inhibitor | Bimake | B14002 | |
| SpeedVac vacuum concentrator | Eppendorf | Concentrator plus | |
| Tabletop ultracentrifuge | Beckman Coulter | Optima MAX-XP | Ultracentrifuge rotor (TLA 55) |
| Transmission electron microscope | HITACHI | H-7650B | |
| TSG101 | Sigma | AF8258 | |
| Ultracentrifuge | Beckman Coulter | Optima XPN-100 | Ultracentrifuge rotor (SW32 Ti) |
| Ultrasonic cell disruptor | Scientz | SCIENTZ-IID | |
| Western Blot imager | Bio-Rad | ChemiDocXRs | Image lab 4.0 (beta 7) |
| β-actin | Sigma | A3853 |
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Cite This Article
Cheng, J., Zhu, J., Liu, Y., Yang, C., Zhang, Y., Liu, Y., Jin, C., Wang, J. Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages. J. Vis. Exp. (196), e65521, doi:10.3791/65521 (2023).