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。

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。
<ol><li>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。</li>
	<li>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培养基（见 材料表）。</li>
	<li>在收集 sEV 之前，请丢弃培养基。用PBS（磷酸盐缓冲盐水; 目录）并用含有10%不含sEVs的FBS的培养基代替。</li>
	<li>根据实验需要，收集细胞上清液并将其转移到50 mL离心管中。</li>
	<li>将细胞上清液以300× g 离心10分钟以除去细胞，弃去沉淀，并将上清液转移到新的50mL离心管中。</li>
	<li>将上清液以2000 ×g 离心10分钟以除去死细胞，弃去沉淀，并将上清液转移到新的高速离心管中（见 材料表）。</li>
	<li>通过高速离心机将上清液以10,000× g 离心30分钟以除去细胞碎片和微泡，弃去沉淀，并将上清液转移到新的超速离心管中（参见 材料表）。开放式超速离心管的体积为38.6 mL;在每个试管中放置 35 mL 细胞上清液。</li>
	<li>将上清液在水平吊篮转子离心机中以110,000×g离心70分钟，以获得粗sEV沉淀。</li>
	<li>弃去上清液，并用 1 mL PBS 洗涤富含 sEVs 的沉淀。在台式超速离心机上以110,000 × g 离心70分钟（见 材料表）。弃去上清液，向超速离心管中加入 1 mL PBS。</li>
	<li>然后连续移液沉淀，将1mL的PBS转移到另一个超速离心管中，依此类推，直到所有超速离心管通过移液混合。</li>
	<li>弃去上清液，并将沉淀重悬于100μL的PBS（即sEV）中。</li>
	<li>使用二辛可宁酸（BCA）方法测量sEV的总蛋白质，按照以下步骤操作（参见 材料表）。<ol><li>蛋白质标准品的制备：将1.2 mL蛋白质制备溶液加入标准蛋白质管（30 mg BSA）中使其完全溶解，以制备蛋白质标准溶液（25 mg / mL）。然后用PBS稀释至终浓度为0.5mg / mL。</li>
		<li>将 0 μL、1 μL、2 μL、4 μL、8 μL、12 μL、16 μL 和 20 μL 蛋白质标准品加入 96 孔板中，并加入 PBS 以弥补 20 μL。同时，向待测样品孔中加入 18 μL HEPES 裂解缓冲液（20 mM HEPES、50 mM NaCl、1 mM NaF、0.5% Triton X-100）和 2 μL sEVs 样品。</li>
		<li>按照制造商的说明制备所需的BCA工作溶液，向每个孔中加入200μLBCA工作溶液，并在室温（RT）下放置20-30分钟。</li>
		<li>用酶标仪测量562nm波长处的吸光度，并根据标准曲线和稀释因子计算sEV的总蛋白质浓度。 注意：通过本协议从40 mL上清液中提取的sEV可用于随后鉴定蛋白质标志物（蛋白质印迹）。然而，从 200 mL 细胞上清液中提取的 sEV 可用于形态学观察（透射电子显微镜，TEM）和粒度分析（纳米颗粒跟踪分析，NTA）。具体来说，对于在步骤1.11中收获的sEV，用100μLPBS重悬。取 20-30 μL 进行形态观察 （TEM），并将剩余样品重悬于 1 mL PBS 中以进行粒度分析 （NTA）。所有离心机都提前预冷。对于步骤1.8，这些超速离心管必须严格平衡并至少装满四分之三。如果没有，请在化妆品中添加介质。对于步骤1.11，sEV可以在4°C下短期储存（12小时）;否则，它们必须储存在-20或-80°C，以避免蛋白质降解干扰肽的提取。但是，对于用于观察形态和测量粒度的样品，仅建议在4°C下短期储存（12小时）。</li>
	</ol></li>
</ol>2. 透射电子显微镜观察sEV的形貌
<ol><li>将一滴新鲜的sEVs样品（约20-30μL）放在封口膜上，将铜网的数面放在sEV液滴上，让它吸收3分钟。</li>
	<li>用滤纸吸收多余的液体，然后将铜网格放在一滴蒸馏水上并洗涤两次。</li>
	<li>用滤纸吸收多余的液体。然后将铜网格放在0.5%乙酸铀酰液滴上进行负染色5秒。</li>
	<li>重复步骤 2.2。</li>
	<li>将准备好的铜网放在样品棒上并将其插入样品台。<ol><li>将放大倍率调整为20,000x-25,000x以找到要测试的样品。然后将放大倍率调整到100,000倍，并调整适当的位置和灰度，以便在透射电子显微镜（TEM; 目录）。用于采集图像的加速电压设置为80 kV。 注意：对于步骤2.1，如果sEVs样品的浓度较低（<50μg/ μL），则吸附时间可以延长至5分钟甚至更长。或者，对于步骤1.10，可以使用较小体积的PBS（50μL）进行重悬。对于步骤2.3，负染色时间不宜过长;否则，很难观察到sEV的三维（3D）结构。</li>
	</ol></li>
</ol>3. 通过纳米颗粒跟踪分析测量sEVs的粒度分布和浓度
<ol><li>对于在步骤1.11中收获的sEV，将溶液稀释至1 mL进行纳米颗粒跟踪分析（NTA）。首先，用蒸馏水清洁仪器，直到视野中没有杂质。然后使用1 mL无菌注射器缓慢推动样品（进样体积：0.5-1 mL）。</li>
	<li>通过支持软件分析样品（见 材料表）。具体来说，单击 启动 相机并将 相机级别 调整为适当的大小（一般强度为 14-16 个单位），然后选择测量方法， 标准测量 （3 或 5 次），然后单击 运行 以收集颗粒。</li>
	<li>收集粒子后，计算机可以观察移动的sEV粒子。同时，设置 检测阈值 （一般为5）来分析粒度分布，使最终计数的颗粒都是移动的sEV而不是背景。分析颗粒后，保存并导出分析结果。</li>
	<li>使用后，用蒸馏水清洗仪器，直到视野中没有明显的颗粒，然后关闭激光。 注意：与用于 TEM 的 sEV 样品不同，用于 NTA 的 sEV 样品不应太浓缩，并且需要更大的样品体积（至少 1 mL）。此外，还可以使用替代方法来分析sEV的尺寸分布，例如流式细胞术和可调电阻脉冲传感（TRPS）等。NTA测量（NanoSight LM10）的推荐粒子/帧数为40。</li>
</ol>4. 蛋白质印迹法检测sEVs的蛋白质标志物
注意：根据细胞外囊泡研究的最小信息（MISEV）2018指南17,18，推荐使用5类蛋白质来表征sEV。评估每个类别 1 至 4 的至少一种蛋白质标记物用于 sEV 制备。
<ol><li>对于在步骤1.9中收获的sEV，小心移取上清液，并加入15μLHEPES裂解缓冲液5分钟。然后加入等量的上样缓冲液，在沸水中煮15分钟。</li>
	<li>在该协议中，sEVs蛋白质的负载量为8-10μg。在10%凝胶中以80V的恒定电压（约30分钟）电泳蛋白质。<ol><li>当样品进入分离凝胶时，将电压调节至120 V（约45分钟）。样品距离玻璃板底部2-3厘米后，断开电源并切断样品条进行电穿孔。电泳溶液的组成在子步骤4.2.1.1中描述。<ol><li>要制备 5 x 电泳溶液，分别称取 15.1 g、5 g 和 94 g 的 Tris、SDS 和甘氨酸，并用去离子水稀释至 1 L。使用时，需要用水稀释5倍。</li>
		</ol></li>
	</ol></li>
	<li>组装电穿孔装置并放入足够的电穿孔溶液（约1L），并在冰上以90mA的恒定电流将蛋白质电穿孔在硝酸纤维素（NC）膜上3.5小时。 注意：要制备10x电穿孔溶液，分别称取30.25g和144.1g的Tris和甘氨酸，并用去离子水稀释至1L。使用时，需要以7：2：1的比例制备去离子水：甲醇：电穿孔溶液。</li>
	<li>电穿孔后，将NC膜置于封闭溶液（2.5g脱脂奶粉在50mLTris缓冲盐水中，0.1%吐温20（TBST）在室温下或4°C下过夜1-2小时。 TBST的组成在子步骤4.4.1中描述。<ol><li>要制备 10x TBST，分别称取 60.56 g 和 175.32 g 的 Tris 和 NaCl，然后加入 10 mL 吐温-20 和 34 mL 浓盐酸。用浓盐酸调节pH = 7.6，并用去离子水补充至2L。使用时，需要用水稀释十倍。</li>
	</ol></li>
	<li>通过三个sEV标记（分化簇9[CD9];β-肌动蛋白;肿瘤易感基因[TSG101]）和一个非sEV标记（葡萄糖调节蛋白94[GRP94]）鉴定蛋白质标记物。<ol><li>移液2μL上述抗体并以1：500稀释，然后将NC膜在室温下孵育3小时或在4°C下孵育过夜。 稀释剂是步骤4.4中的封闭溶液。</li>
		<li>与一抗孵育后，在摇床上用1x TBST洗涤3次，每次10分钟。</li>
	</ol></li>
	<li>将NC膜置于稀释的二抗（1：500）中，并在室温振荡器上缓慢摇动45-50分钟。用1x TBST在摇床上洗涤3次，每次10分钟。</li>
	<li>以1：1混合化学发光底物A和B（参见材料表），将混合试剂加入NC膜中，并在室温下孵育5分钟。</li>
	<li>使用印迹成像仪对NC膜进行成像（参见 材料表）<ol><li>打开 图像实验室 软件，然后选择 新建实验方案。</li>
		<li>选择印 迹比色应用，取消 突出显示，单击 运行实验方案，获取标记物并保存。</li>
		<li>然后重新选择应用程序印 迹化学，并将 图像总数 和 曝光时间 分别设置为30秒和300秒。</li>
		<li>单击 运行实验方案，获取图像并保存。最后，拆下NC膜并关闭计算机。 注意：对于步骤4.6，二抗的孵育时间不应超过50分钟;否则，背景会太暗。</li>
	</ol></li>
</ol>5. sEVs肽的提取
<ol><li>通过在4°C下以110,000× g 超速离心70分钟洗涤sEV两次，然后加入500μLPBS（参见 材料表）以重悬它们，并加入5μL的100x蛋白酶抑制剂（参见 材料表）。</li>
	<li>放置样品进行超声波破碎（见 材料表）。超声波均质的设置如下：功率：40 W，总时间：10 分钟，超声波时间：3 秒，间隔时间：5 秒。</li>
	<li>将粉碎的混合物在4°C下以13,700 ×g 离心15分钟。</li>
	<li>离心后，加入二硫苏糖醇（DTT; 目录）至上清液至终浓度为50mmol/L，并在56°C的水浴中孵育30分钟。</li>
	<li>随后，加入 50 μL 碘乙酰胺 （IAA; 目录）到浓度为1mol/L的上清液中，并在黑暗中在室温下反应20分钟。</li>
	<li>将混合物在4°C下以13,700× g 离心15分钟，并收集上清液，粗肽提取物。将其储存在-20°C以备后用。</li>
	<li>用10kDa超滤管19（见 材料表）超滤所得的肽粗提取物以除去蛋白质，并在4°C下以13,700 ×g 离心1小时。收集流出物并在45°C的真空离心浓缩器（见 材料表）中干燥。</li>
	<li>按照步骤5.8.1-5.8.8对干燥的样品进行脱盐。使用质谱级纯水作为试剂的溶剂。<ol><li>向每个样品中加入 100 μL 0.1% 三氟乙酸 （TFA），以完全溶解干燥的肽。</li>
		<li>向每根脱盐柱中加入 100 μL 100% 乙腈 （ACN），在室温下以 400 × g 离心 3 分钟，然后弃去流出物。然后加入 100 μL 50% 乙腈，以 400 × g 离心 3 分钟，弃去流出物。</li>
		<li>向每根脱盐柱中加入 100 μL 0.1% TFA，以 400 × g 离心 3 分钟，然后弃去流出物。</li>
		<li>重复步骤 5.8.3。</li>
		<li>将步骤5.8.1中溶解的肽移液到脱盐柱中，以400× g 离心3分钟，并回收流出物。重复样品上样步骤，让肽样品吸附到脱盐柱中。</li>
		<li>重复步骤 5.8.3 两次。</li>
		<li>向脱盐柱中加入 50 μL 50% 乙腈（含 0.1% TFA），以 400 × g 离心 3 分钟，并将流出物（肽）收集在新的质谱级离心管中。</li>
	</ol></li>
</ol>6. 干燥脱盐肽
<ol><li>在真空离心浓缩器中干燥脱盐肽（设置： V-AQ 模式，温度：45°C，时间：50分钟），并将其用于随后的质谱分析。 注意：sEVs肽的提取过程应尽可能在冰上进行，以避免蛋白质降解。所有使用的试剂均用质谱级水制备，以避免塑料污染。对于步骤5.8，脱盐导致肽样品不可避免的损失。通过重复步骤5.8.5和5.8.7可以减少这种损失。</li>
</ol>7. 液相色谱-串联质谱（LC-MS/MS）分析
注意：通过液相色谱-串联质谱（LC-MS/MS）分析肽样品，特别是与EASY-nLC 1000纳米高性能LC系统连接的Orbitrap Q Exactive HF-X质谱仪（参见 材料表）。
<ol><li>在C18分析柱（1.9μm晶粒直径，15cm长x 150μm内径）上以60nL / min的流速以65分钟梯度分离样品：4%-15%4分钟，15%-28%28分钟，28%-40%10分钟，40%-69%10分钟，95%恒定7分钟， 95%降至6%1分钟，恒定5分钟（溶剂A，含0.1%甲酸[FA]的水;溶剂B，含0.1%FA，v/v的80%乙腈）。</li>
	<li>在纳米电喷雾电离（nano-ESI）喷雾器中，在320°C毛细管温度和2.2kV的喷雾电压下电离洗脱肽。</li>
	<li>使用数据依赖性采集模式收集质谱数据，全扫描模式下的分辨能力为 120,000，MS /MS 模式下的分辨能力为 15,000。<ol><li>在 250 m/z 至 1800 m/z 的轨道上执行全面扫描，隔离窗口为 1.6 m/z。</li>
		<li>选择前 20 个强离子进行高能碰撞解离 （HCD） 碎裂，归一化碰撞能量为 29%，并在离子分离器中测量。 注意：典型的质谱条件如下：自动增益控制（AGC）目标为3 x 106 离子（全扫描）和2 x 105 离子（MS/MS扫描）;全扫描的最长注入时间为80 ms，MS / MS扫描的最长注入时间为19 ms;动态排除使用13 s。</li>
	</ol></li>
</ol>

分离小的细胞外囊泡并提取其肽组

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W., Zhu, H. J., Takahashi, N., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+isolation+and+characterization:+toward+clinical+application.">Extracellular vesicle isolation and characterization: toward clinical application.</a> The Journal of Clinical Investigation. 126 (4), 1152-1162 (2016).</li><li>Li, P., Kaslan, M., Lee, S. H., Yao, J., Gao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+exosome+isolation+techniques.">Progress in exosome isolation techniques.</a> Theranostics. 7 (3), 789-804 (2017).</li><li>Palanski, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+urine+peptidomics+workflow+identifies+chemically+defined+dietary+gluten+peptides+from+patients+with+celiac+disease.">An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease.</a> Nature Communications. 13, 888 (2022).</li><li>Kalaora, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+bacteria-derived+HLA-bound+peptides+in+melanoma.">Identification of bacteria-derived HLA-bound peptides in melanoma.</a> Nature. 592 (7852), 138-143 (2021).</li><li>Hamley, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+bioactive+peptides+for+biomaterials+design+and+therapeutics.">Small bioactive peptides for biomaterials design and therapeutics.</a> Chemical Reviews. 117 (24), 14015-14041 (2017).</li><li>Lotvall, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+experimental+requirements+for+definition+of+extracellular+vesicles+and+their+functions:+a+position+statement+from+the+International+Society+for+Extracellular+Vesicles.">Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.</a> Journal of Extracellular Vesicles. 3, 26913 (2014).</li><li>Thery, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Kim, Y. G., Lone, A. M., Saghatelian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+proteolysis+of+bioactive+peptides+using+a+peptidomics+approach.">Analysis of the proteolysis of bioactive peptides using a peptidomics approach.</a> Nature Protocols. 8 (9), 1730-1742 (2013).</li><li>Lyapina, I., Ivanov, V., Fesenko, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidome:+Chaos+or+inevitability.">Peptidome: Chaos or inevitability.</a> International Journal of Molecular Sciences. 22 (23), 13128 (2021).</li><li>Keller, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoy+exosomes+provide+protection+against+bacterial+toxins.">Decoy exosomes provide protection against bacterial toxins.</a> Nature. 579 (7798), 260-264 (2020).</li><li>Koeppen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Let-7b-5p+in+vesicles+secreted+by+human+airway+cells+reduces+biofilm+formation+and+increases+antibiotic+sensitivity+of+P.+aeruginosa.">Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (28), e2105370118 (2021).</li></ol>

提交人声明，他们没有相互竞争的经济利益。

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Beyotime Technology</td>
			<td>P0012</td>
			<td></td>
		</tr>
		<tr>
			<td>CD9</td>
			<td>Beyotime Technology</td>
			<td>AF1192</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal filter tube</td>
			<td>Millipore</td>
			<td>UFC5010BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge bottles polypropylene</td>
			<td>Beckman Coulter</td>
			<td>357003</td>
			<td>High-speed centrifuge</td>
		</tr>
		<tr>
			<td>Chemiluminescent substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34580</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Solarbio</td>
			<td>D8220</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>DMEM culture medium</td>
			<td>Cell World</td>
			<td>N?A</td>
			<td></td>
		</tr>
		<tr>
			<td>GRP94</td>
			<td>Cell Signaling Technology</td>
			<td>20292</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Avanti JXN-26</td>
			<td>Centrifuge rotor (JA-25.50)</td>
		</tr>
		<tr>
			<td>Immortalized bone marrow-derived macrophages (iBMDM)</td>
			<td>National Institute of Biological Sciences, China</td>
			<td></td>
			<td>Provided by Dr. Feng Shao (National Institute of Biological Sciences, China)</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>l1149</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Microfuge tube polypropylene</td>
			<td>Beckman Coulter</td>
			<td>357448</td>
			<td>1.5 mL, Tabletop ultracentrifuge&#160;</td>
		</tr>
		<tr>
			<td>nano-high-performance LC system</td>
			<td>Thermo Fisher Scientific</td>
			<td>EASY-nLC 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle tracking analysis&#160;</td>
			<td>Malvern Panalytical</td>
			<td>NanoSight LM10</td>
			<td>NanoSight NTA3.4</td>
		</tr>
		<tr>
			<td>Orbitrap Q Exactive HF-X mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Solarbio</td>
			<td>P1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyallomer centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td>Ultracentrifuge</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Bimake</td>
			<td>B14002</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac vacuum concentrator</td>
			<td>Eppendorf</td>
			<td>Concentrator plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima MAX-XP</td>
			<td>Ultracentrifuge rotor (TLA 55)</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>HITACHI</td>
			<td>H-7650B</td>
			<td></td>
		</tr>
		<tr>
			<td>TSG101</td>
			<td>Sigma</td>
			<td>AF8258</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima XPN-100</td>
			<td>Ultracentrifuge rotor (SW32 Ti)</td>
		</tr>
		<tr>
			<td>Ultrasonic cell disruptor</td>
			<td>Scientz</td>
			<td>SCIENTZ-IID</td>
			<td></td>
		</tr>
		<tr>
			<td>Western Blot imager</td>
			<td>Bio-Rad</td>
			<td>ChemiDocXRs</td>
			<td>Image lab 4.0 (beta 7)</td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Sigma</td>
			<td>A3853</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of peptides of small extracellular vesicles from bone marrow-derived macrophages

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。

Small extracellular vesicles (sEVs) with a diameter of less than 200 nm are present in almost all types of body fluids and secreted by all kinds of cells, including urine, sweat, tears, cerebrospinal fluid, and amniotic fluid1. Initially, sEVs were considered as receptacles for disposing of cellular waste, which led to minimal research in the subsequent decade2. Recently, increasing evidence indicates that sEVs contain specific proteins, lipids, nucleic acids, and other metabolites. These molecules are transported to target cells3, contributing to intercellular communication, through which they participate in various biological processes, such as tissue repair, angiogenesis, immunity4 and inflammation5,6, tumor development and metastasis7,8,9, etc.
To facilitate the study of sEVs, it is imperative to isolate sEVs from complex samples. Different sEVs isolation methods have been developed based on the physical and chemical properties of sEVs, such as their density, particle size, and surface marker proteins. These techniques include ultracentrifugation-based methods, particle size-based methods, immunoaffinity capture-based methods, sEVs precipitation-based methods, and microfluidics-based methods10,11,12. Among these techniques, the ultracentrifugation-based method is widely recognized as the gold standard for sEVs isolation and is the most commonly used technique13.
An increasing amount of evidence suggests the presence of a multitude of undiscovered biologically active peptides in the peptidomes of various organisms. These peptides significantly contribute to numerous physiological processes by regulating growth, development, stress response14,15, and signal transduction16. The objective of sEVs&#39; peptidome is to uncover the peptides carried by these sEVs and provide clues to their biological functions. Here, we present a protocol of isolating sEVs through differential ultracentrifugation, followed by extraction of peptides from these sEVs for further analysis of their peptidome.

作者聚焦： 从骨髓来源的巨噬细胞中分离的小细胞外囊泡中提取肽组 

从骨髓来源的巨噬细胞中鉴定小细胞外囊泡的肽

We explore the functional roles of extracellular vesicles in innate immunity. We have developed a protocol to isolate small extracellular vesicles, or sEVs, via differential ultracentrifugation and identify the peptidome by LC-MS/MS, revealing the key biological functions of the peptides in sEVs. The extracellular vesicles have been shown to mediate intercellular communication by transporting cargo to recipient cells, such as proteins, lipids, and nucleic acids.By releasing antimicrobial components, they act the first line of defense against invading microbes in innate immunity. While differential ultracentrifugation yields highly pure small extracellular sEVs, it can be time-consuming and often results in low yields. In our study, these are major challenges for the stable identification of low-abundance peptides in the sEVs.

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...

Watch this Scientific Journal Video about Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages at JoVE.com

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。

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of ...

我们探索细胞外囊泡在先天免疫中的功能作用。我们开发了一种通过差示超速离心分离小细胞外囊泡（sEV）的方案，并通过LC-MS / MS鉴定肽球，揭示了sEV中肽的关键生物学功能。细胞外囊泡已被证明可以通过将货物运输到受体细胞（如蛋白质、脂质和核酸）来介导细胞间通讯。通过释放抗菌成分，它们在先天免疫中充当抵御入侵微生物的第一道防线。虽然差示超速离心可产生高纯度的小细胞外sEV，但它可能非常耗时，并且通常会导致低产量。在我们的研究中，这些是稳定鉴定sEV中低丰度肽的主要挑战。

identification-peptides-small-extracellular-vesicles-from-bone-marrow

Research

JoVE Journal

Biochemistry

Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages

1.2K 次观看.  Anhui Medical University. 我们探索细胞外囊泡在先天免疫中的功能作用。我们开发了一种通过差示超速离心分离小细胞外囊泡（sEV）的方案，并通过LC-MS / MS鉴定肽球，揭示了sEV中肽的关键生物学功能。细胞外囊泡已被证明可以通过将货物运输到受体细胞（如蛋白质、脂质和核酸）来介导细胞间通讯。通过释放抗菌成分，它们在先天免疫中充当抵御入侵微生物的第一道防线。虽然差示超速离心可...

视频: 作者聚焦： 从骨髓来源的巨噬细胞中分离的小细胞外囊泡中提取肽组 

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of &lt;200 nm are present in various body fluids. These sEVs regulate various biological processes such as gene transcription and translation, cell proliferation and survival, immunity and inflammation through their cargos, such as proteins, DNA, RNA, and metabolites. Currently, various techniques have been developed for sEVs isolation. Among them, the ultracentrifugation-based method is considered the gold standard and is widely used for sEVs isolation. The peptides are naturally biomacromolecules with less than 50 amino acids in length. These peptides participate in a variety of biological processes with biological activity, such as hormones, neurotransmitters, and cell growth factors. The peptidome is intended to systematically analyze endogenous peptides in specific biological samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we introduced a protocol to isolate sEVs by differential ultracentrifugation and extracted peptidome for identification by LC-MS/MS. This method identified hundreds of sEVs-derived peptides from bone marrow-derived macrophages.

This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.

科研

生物化学

Isolating Small Extracellular Vesicles (sEVs) by Differential Ultracentrifugation

Isolating Small Extracellular Vesicles (SEVs) by Differential Ultracentrifugation  

To begin, centrifuge FBS overnight in an ultracentrifuge at 110, 000 G and four degrees Celsius to remove the endogenous sEVs. Sterilize the supernatant by filtering it through a 0.2 micron ultrafiltration membrane to yield sEVs-free FBS. Next in a 150 millimeter culture dish, plate about 3 times 10 to the seventh immortalized bone-marrow-derived macrophages in 20 milliliters of DMEM culture medium.Incubate the dish at 37 degrees Celsius under 5%carbon dioxide overnight prior to collecting the sEVs from the macrophages. The next day, after discarding the medium, wash the cells with phosphate-buffered saline or PBS. Replace the medium with DMEM containing 10%sEVs-free free fetal bovine serum before incubating the cells at 37 degrees Celsius and 5%carbon dioxide.Based on the experiment's requirements, collect and transfer the cells'supernatant to 50 milliliter centrifuge tubes. Centrifuge the tubes at 300 G for 10 minutes to remove the cells. Transfer the result in supernatant from each tube to a new 50 milliliter centrifuge tube and discard the pellet.This time, centrifuge the supernatant at 2000 G for 10 minutes to remove dead cells. Then transfer the supernatant to new high-speed centrifuge tubes and discard the pellets. Next to remove debris and microvesicles, centrifuge the obtained supernatant at 10, 000 G for 30 minutes using a high-speed centrifuge.Transfer the resulting supernatant to new ultracentrifuge tubes by adding 35 milliliters in each tube and discarding the pellets. Centrifuge the ultracentrifuge tubes in a swinging bucket rotor centrifuge at 110, 000 G for 70 minutes before discarding the supernatant. Wash the resulting crude sEVs-enriched pellet with one milliliter of PBS.Again, add one milliliter of PBS to the washed pellet in one of the tubes, and mix by pipetting continuously. Transfer the one milliliter of resultant PBS suspension to another tube containing the pellet, and repeat the same until all pellets in the tubes are mixed by pipetting. Transfer the resultant one milliliter of sEVs-rich PBS to a new ultracentrifuge tube.Then centrifuge the new tube in a tabletop ultracentrifuge at 110, 000 G for 70 minutes. After discarding the supernatant, wash the resulting crude sEVs-enriched pellet with 100 microliters of PBS. Add 1.2 milliliters of protein preparation solution in a standard protein tube to dissolve the 30 milligrams of BSA present in it.Dilute the resulting protein standard solution with PBS to reduce its concentration from 25 to 0.5 milligrams per milliliter. Add eight different volumes of the diluted protein standard solution into wells of a 96-well plate, and bring the volume to 20 microliters in each well using PBB. Add 18 microliters of HEPES lysis buffer to the sample wells, followed by the addition of two microliters of the sEVs samples.Next, add 200 microliters of BCA working solution prepared per the manufacturer's instructions into each well. Allow the plate to rest at room temperature for 20 to 30 minutes. Finally, measure the absorbance at 562 nanometers using a microplate reader.Calculate the total protein content in the sEVs using the obtained results. The transmission electron microscopy images of the isolated sEVs displayed a typical cup-like morphology. Nanoparticle tracking analysis showed that the isolated sEVs were mostly concentrated at 136 nanometers.Western blot showed that isolated sEVs were significantly enriched of sEVs markers, including CD9, beta-actin, and TSG101. The endoplasmic reticulate marker GRP94 was only detected in the whole-cell lysate. The results indicated that the method employed yielded sEVs with a high level of purity.

通过差示超速离心分离小细胞外囊泡（sEV）

Extraction of Peptides from Isolated Small Extracellular Vesicles (sEVs)

To begin, wash the isolated small extracellular vesicles or SEVs twice by ultracentrifugation at 110, 000 G and four degrees Celsius for 70 minutes. Re-suspend the vesicles in 500 microliters of phosphate buffered saline or PBS. Then, add five microliters of 100X protease inhibitor to it.Subject the sample to ultrasonic crushing at 40 watts for 10 minutes, with three second ultrasonic time, and five second interval time. Centrifuge the crushed mixture at 13, 700 G for 15 minutes at four degrees Celsius. To the obtained supernatant, add dithiothreitol to a final concentration of 50 millimolar.And incubate it in a water bath at 56 degrees Celsius for 30 minutes. Then add 50 microliters of one molar iodoacetamide to the supernatant. And allow it to react at room temperature for 20 minutes in the dark.Centrifuge the mixture at 13, 700 G and four degrees Celsius for 15 minutes. And transfer the supernatant containing the crude peptide extract to a 10 kilodalton ultracentrifuge tube. Remove protein from the crude peptide by centrifuging the ultra filtration tube at 13, 700 G and four degrees Celsius for one hour.Dry the collected effluent in a vacuum centrifugal concentrator at 45 degrees Celsius. For desalting, use mass spectrometry grade pure water as a solvent for all reagents. First, dissolve the dry peptides in 100 microliters of 0.1%trifluoracetic acid or TFA, and keep it aside.Then add 100 microliters of pure acetonitrile to each desalting column. Centrifuge the columns at 400 G for three minutes at room temperature, and discard the effluent. Then add 100 microliters of 50%acetonitrile per desalting column, and repeat the centrifugation as mentioned previously before discarding the effluent.Next, add 100 microliters of 0.1%TFA to each desalting column before centrifuging as mentioned previously. Once again, discard the effluent. Now, pipet the desalt peptides into the desalting column.Centrifuge the columns as mentioned previously to load the sample. Add the recovered effluent back to the desalting column to repeat the loading. Then, add 100 microliters of 0.1%TFA.Centrifuge as mentioned previously, and discard the effluent. Finally, add 50 microliters of 50%acetonitrile containing 0.1%TFA to the peptide in the desalting column. After centrifusion, as mentioned previously, collect the effluent in a new mass spectrometry grade centrifuge tube.

作者聚焦： 从骨髓来源的巨噬细胞中分离的小细胞外囊泡中提取肽组

作者聚焦：从骨髓来源的巨噬细胞中分离的小细胞外囊泡中提取肽组