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>

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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。

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

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 

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。

从骨髓来源的巨噬细胞中鉴定小细胞外囊泡的肽

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

1.9K 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 

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

组织细胞外囊泡与肝脏分离

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

基于荧光的人体血液单细胞外囊快速表征

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5&#39;-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

使用MALDI-TOF质谱法鉴定酶降解产生的RNA片段

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

小细胞外囊泡高通量分离纯化的流式细胞术分选参数优化

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

增强 EV 分离以进行精确的生物流体分析

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 cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

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 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

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

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