这项工作部分由 NIH 赠款 3RF1AG064250 和 R44CA239845资助。

所有尿液样本均在知情同意后从健康个体中收集。这些实验符合涉及人体样本的所有伦理标准，并符合普渡大学人类研究保护计划的指导方针。
1. 样品采集
<ol><li>在2,500× g，4°C下将12mL尿液样品在15mL锥形离心管中离心10分钟，以除去细胞碎片和大凋亡体。</li>
	<li>将 10 mL 上清液转移到新的 15 mL 试管中，然后进行 EV 分离。 注意：方案可以在这里暂停，样品可以储存在-80°C，并在使用时在37°C解冻。</li>
</ol>2. 使用EVtrap方法进行EV隔离
<ol><li>根据制造商的说明，向样品中加入 0.5 mL 上样缓冲液（1：10 v/v 比）和 100 μL EVtrap 珠浆（1：50 v/v 比）。</li>
	<li>在室温下通过端到端旋转孵育样品30分钟。</li>
	<li>通过将 15 mL 锥形管放在磁性分离器架上来沉淀样品。除去上清液。</li>
	<li>将珠子重悬于 1 mL 洗涤缓冲液中，并将悬浮液转移到 1.5 mL 微量离心管中。轻轻移液以重新悬浮与 EV 结合的磁珠。</li>
	<li>将试管放在 1.5 mL 微量离心管磁性分离器架上并吸出上清液。使用P200移液管完全吸出上清液并避免吸出珠子。</li>
	<li>用 1 mL 洗涤缓冲液洗涤珠子。吸出上清液后立即加入缓冲液，以防止珠子变干。</li>
	<li>在室温下用 1 mL 磷酸盐缓冲盐水 （PBS） 洗涤珠子 2 次。</li>
	<li>在室温下用 100 μL 新鲜制备的 100 mM 三乙胺孵育珠子 10 分钟，并使用 1.5 mL 微量离心管磁分隔架收集含有 EV 的洗脱溶液。 注：要制备 1 mL 的 100 mM 三乙胺，请在水中稀释 14 μL 三乙胺溶液。</li>
	<li>重复步骤2.8并合并洗脱的溶液。使用真空离心浓缩器在4°C下干燥洗脱液。 注意：实验可以在此处暂停。干燥的EV样品可以在-80°C下储存数月，而不会对以下步骤或结果产生不利影响。</li>
</ol>3. 通过蛋白质印迹法表征 EV
<ol><li>将 5% 的干燥 EV 样品（相当于 0.5 mL 尿液样品）重悬于 20 μL 含有 10 mM 二硫苏糖醇 （DTT） 的 1x 十二烷基硫酸锂 （LDS） 样品缓冲液中。 注意：来自 0.5 mL 尿液的 EV 足以检测蛋白质印迹中的 CD9（EV 标志物24）信号。</li>
	<li>将样品在95°C下煮沸5分钟。 将样品加载到聚丙烯酰胺凝胶上，并按照标准方案25进行电泳和免疫印迹。</li>
	<li>将蛋白质转移到低荧光聚偏二氟乙烯（PVDF）膜上后，在室温下用1%牛血清白蛋白（BSA）在tris缓冲盐水吐温-20（TBST）中封闭膜1小时。要制备 1 L TBST 缓冲液，加入 19 mM Tris 碱、137 mM NaCl 和 1 mL 吐温-20;使用 HCl 将 pH 值调节至 7.4。</li>
	<li>将膜与兔抗CD9抗体以1：5,000的比例在TBST中的1%BSA中在4°C下孵育过夜或在室温下孵育2小时。</li>
	<li>用TBST洗涤膜3次，每次5分钟。在室温下，将膜与抗兔IgG，HRP连接的二抗在TBST中的1%BSA中以1：5000的比例孵育1小时。</li>
	<li>用TBST洗涤膜3次，每次5分钟。将市售的增强型化学发光（ECL）底物（比例为1：1）添加到膜上，并在化学发光成像系统上检测信号。</li>
</ol>4. 蛋白质组学和磷酸化蛋白质组学分析的样品制备
<ol><li>制备含有 12 mM 脱氧胆酸钠 （SDC）、12 mM 月桂酰肌氨酸钠 （SLS）、100 mM 三乙基碳酸氢铵缓冲液 （TEAB）、10 mM 三（2-羧乙基）膦 （TCEP）、40 mM 氯乙酰胺 （CAA） 和 1x 磷酸酶抑制剂混合物的新鲜裂解缓冲液。 注意：库存溶液列在 材料表的描述中。裂解缓冲液的制备方法是加入储备溶液，以达到所需的浓度，具体取决于所需的体积。</li>
	<li>将干燥的EV样品溶解在100μL裂解缓冲液中，并在95°C下加热样品10分钟，并以1,100rpm振荡。</li>
	<li>将样品冷却至室温后，加入 400 μL 50 mM TEAB，将其稀释五倍。</li>
	<li>按照制造商的说明使用BCA检测试剂盒测量蛋白质浓度。使用用 50 mM TEAB 稀释 5 倍的裂解缓冲液作为空白。</li>
	<li>加入胰蛋白酶/Lys-C混合物，酶蛋白比为1：50 w/w，并将样品在37°C下孵育过夜，以1,100rpm振荡。</li>
	<li>加入 50 μL 10% 三氟乙酸 （TFA） 酸化样品。</li>
	<li>向样品中加入 600 μL 乙酸乙酯，并将混合物涡旋 2 分钟。</li>
	<li>将样品以20,000× g 离心3分钟，并除去上层（有机层）。避免在抽吸过程中干扰界面。</li>
	<li>重复步骤 4.7-4.8。使用真空离心浓缩器干燥水相。</li>
	<li>将干燥的样品重悬于 200 μL 0.1% TFA 中以酸化肽，并根据制造商的说明使用 C18 脱盐尖端对样品进行脱盐。用 200 μL 0.1% TFA 的 80% 乙腈溶液调节吸头，然后用 200 μL 的 0.1% TFA 调节 2 次。将酸化的肽样品加载到吸头中，然后用 200 μL 0.1% TFA 洗涤吸头 3 次。用 200 μL 0.1% TFA 的 80% 乙腈溶液洗脱肽。</li>
	<li>使用真空离心浓缩器干燥洗脱液。分别干燥 2% 的肽样品（相当于 0.2 mL 尿液样品）用于蛋白质组学分析。使用样品的其余部分（98%）进行磷酸化蛋白质组学分析。</li>
	<li>根据制造商的说明，使用磷酸肽富集试剂盒从样品中富集磷酸肽。执行下面描述的步骤进行扩充。<ol><li>将干燥的样品重悬于200μL上样缓冲液中。向样品中加入 50 μL 珠子，并在室温下剧烈摇动 20 分钟。将带有珠子的样品加载到烧结的尖端，并以 100 x g 离心 1 分钟。</li>
		<li>用 200 μL 上样缓冲液洗涤吸头，然后洗涤缓冲液 1，然后洗涤缓冲液 2。通过以20×g离心一次2分钟和一次以100×g离心1分钟来执行所有三个洗涤步骤。</li>
		<li>将带有珠子的尖端放入新管中以收集洗脱的磷酸肽。向吸头中加入 50 μL 洗脱缓冲液，以 20 x g 离心一次 2 分钟。 向吸头中再加入 50 μL 洗脱缓冲液，并以 20 x g 离心一次 2 分钟。最后一次以100×g离心1分钟。</li>
	</ol></li>
	<li>使用真空离心浓缩器干燥洗脱的磷酸肽。</li>
</ol>5. LC-MS/MS分析
注：可以使用不同的LC-MS/MS系统/设置和数据采集方法，例如数据相关采集（DDA）。
<ol><li>将干燥的蛋白质组/磷酸化蛋白质组样品重悬于0.1%甲酸（溶剂A）中，并按照制造商的说明加载样品。</li>
	<li>通过液相色谱 （LC） 系统将样品注入捕获的离子淌度飞行时间 MS 中，并使用预设的标准化 Whisper 40 样品/天方法。肽在15cm C18柱（75μm内径，1.9μm粒径）上分离，如 材料表中所述。</li>
	<li>对于蛋白质组学分析，使用平行累积-连续碎裂结合数据非依赖性采集 （dia-PASEF） 采集方法采集数据，每个斜坡的质量范围为 300-1200 m/z 和 0.6-1.50 1/K0，循环时间为 1.38 s。</li>
	<li>对于磷酸化蛋白质组学分析，使用dia-PASEF采集方法采集数据，每个斜坡的质量范围为400-1550 m/z和0.6-1.50 1/K0，循环时间为1.38 s。</li>
	<li>将原始文件加载到蛋白质组学软件中，并使用无库数据独立采集工作流程进行信号提取、鉴定和定量。<ol><li>对于检索设置，使用智 人 数据库、胰蛋白酶/P 酶的特定消化类型、7 个最小肽长、52 个最大肽长、2 个缺失裂解、半胱氨酸位点的脲酰胺甲基作为固定修饰、乙酰蛋白 N 项、蛋氨酸氧化和丝氨酸、苏氨酸和酪氨酸的磷酸化（用于磷酸化蛋白质组学分析）作为变量修饰，5 个作为最大变量修饰。将 PSM、肽和蛋白质组的 FDR 设置为 0.01。 注：此协议中使用了Spectronaut软件。其他DIA数据搜索软件，如DIA-NN和PEAKS也是常用的。如果数据是在 DDA 模式下采集的，则适用 MaxQuant 和 Proteome Discoverer 等软件。</li>
	</ol></li>
</ol>

增强 EV 分离以进行精确的生物流体分析

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+for+relative+quantitation+of+proteins+using+tandem+mass+tags+(TMT)+and+mass+spectrometry+(MS).">Sample preparation for relative quantitation of proteins using tandem mass tags (TMT) and mass spectrometry (MS).</a> Methods Mol Biol. 1741, 135-149 (2018).</li><li>Charles Jacob, H. 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=Identification+of+novel+early+pancreatic+cancer+biomarkers+KIF5B+and+SFRP2+from+&#8220;first+contact&#8221;+interactions+in+the+tumor+microenvironment.">Identification of novel early pancreatic cancer biomarkers KIF5B and SFRP2 from &#8220;first contact&#8221; interactions in the tumor microenvironment.</a> J Exp Clinl Cancer Res. 41 (1), 258 (2022).</li><li>Nunez Lopez, Y. O., 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=Extracellular+vesicle+proteomics+and+phosphoproteomics+identify+pathways+for+increased+risk+in+patients+hospitalized+with+COVID-19+and+type+2+diabetes+mellitus.">Extracellular vesicle proteomics and phosphoproteomics identify pathways for increased risk in patients hospitalized with COVID-19 and type 2 diabetes mellitus.</a> Diabetes Res Clin Pract. 197, 110565 (2023).</li><li>Hinzman, C. P., 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=A+multi-omics+approach+identifies+pancreatic+cancer+cell+extracellular+vesicles+as+mediators+of+the+unfolded+protein+response+in+normal+pancreatic+epithelial+cells.">A multi-omics approach identifies pancreatic cancer cell extracellular vesicles as mediators of the unfolded protein response in normal pancreatic epithelial cells.</a> J Extracell Vesicles. 11 (6), e12232 (2022).</li><li>Kornilov, R., 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=Efficient+ultrafiltration-based+protocol+to+deplete+extracellular+vesicles+from+fetal+bovine+serum.">Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum.</a> J Extracell Vesicles. 7 (1), 1422674 (2018).</li><li>Willms, E., 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=Cells+release+subpopulations+of+exosomes+with+distinct+molecular+and+biological+properties.">Cells release subpopulations of exosomes with distinct molecular and biological properties.</a> Sci Rep. 6, 22519 (2016).</li><li>Searle, B. 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=Generating+high+quality+libraries+for+DIA+MS+with+empirically+corrected+peptide+predictions.">Generating high quality libraries for DIA MS with empirically corrected peptide predictions.</a> Nat Commun. 11 (1), 1548 (2020).</li><li>Skowronek, P., 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=Rapid+and+in-depth+coverage+of+the+(Phospho-)proteome+with+deep+libraries+and+optimal+window+design+for+dia-PASEF.">Rapid and in-depth coverage of the (Phospho-)proteome with deep libraries and optimal window design for dia-PASEF.</a> Mol Cell Proteomics. 21 (9), 100279 (2022).</li></ol>

作者宣布了相互竞争的经济利益。Anton Iliuk 和 W. Andy Tao 是 Tymora Analytical Operations 的联合创始人，该公司开发了 EVtrap 微珠并商业化了 PolyMAC 磷酸肽富集试剂盒。

有效的EV分离是检测EV中低丰度蛋白质和磷蛋白的重要先决条件。尽管开发了许多方法来满足这一需求，但大多数方法仍然存在局限性，例如恢复率差或可重复性低，这阻碍了它们在大规模研究和常规临床环境中的使用。DUC 通常被认为是最常见的 EV 隔离方法，通常应用额外的洗涤步骤来帮助提高目标 EV 的纯度27,28。此过程会导致更繁琐和耗时的 DUC 过程...

细胞外囊泡 （EV） 是由所有类型的细胞分泌的膜包膜纳米颗粒，存在于血液、尿液、唾液等生物流体中1,2,3,4。EV 携带多种生物活性分子，这些分子反映了宿主细胞的生理和病理状态，因此是疾病进展的关键因素 4,5,6。此外，广泛的研究已经确定，基于 EV 的疾病标志物可以在症状出现或肿瘤生理检测之前被识别出来 5,6,7。
磷酸化是细胞信号传导和调节的关键机制。因此，磷蛋白为生物标志物的发现提供了有价值的来源，因为异常的磷酸化事件与失调的细胞信号通路和转移性疾病的发展（如癌症）有关8,9,10。尽管分析磷酸化动力学可以识别疾病特异性磷蛋白特征作为潜在的生物标志物，但磷蛋白的低丰度和动态性质对开发磷蛋白作为生物标志物构成了重大挑战11,12。值得注意的是，封装在 EV 中的低丰度磷蛋白在细胞外环境中受到保护，免受外部酶消化的影响8。因此，EV 和 EV 衍生的磷蛋白为癌症和其他疾病的早期检测中的生物标志物发现提供了理想的来源。
尽管对 EV 中蛋白质磷酸化的分析为了解癌症信号传导和早期疾病诊断提供了宝贵的资源，但缺乏有效的 EV 分离方法是一个主要障碍。EV 隔离通常通过差示超速离心 （DUC）13 实现。然而，这种方法耗时且由于通量低和重现性差而不适合临床意义13,14。替代 EV 分离方法（例如聚合物诱导沉淀15）由于非 EV 蛋白的共沉淀而受到低特异性的限制。基于亲和力的方法，包括基于抗体的亲和力捕获16 和亲和力过滤17，可提供增强的特异性，但由于体积小，回收率相对较低。
为了解决探索 EV 中磷蛋白动力学的问题，我们课题组开发了基于化学亲和力的细胞外囊泡全回收和纯化 （EVtrap） 技术，将 EV 捕获到功能化的磁珠上18。先前的结果表明，与DUC和其他现有分离方法相比，这种基于磁珠的EV分离方法在从各种生物流体样品中分离EV方面非常有效，并且能够实现更高的EV产量，同时最大限度地减少污染18,19。我们已经成功地利用了 EVtrap 和我们小组20 开发的钛基磷酸肽富集方法来分析来自不同生物流体的 EV 的磷酸化蛋白质组，并检测各种疾病的潜在磷蛋白生物标志物19、21、22。
在这里，我们提出了一种基于EVtrap的协议，用于分离循环的EV。该协议侧重于泌尿 EV。我们还演示了使用蛋白质印迹法对分离的 EV 进行表征。然后，我们详细介绍了蛋白质组学和磷酸化蛋白质组学分析的样品制备和质谱 （MS） 采集。该协议为分析尿液 EV 蛋白质组和磷酸化蛋白质组提供了高效且可重复的工作流程，这将有助于对 EV 及其临床应用的进一步研究23。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Life Science Products</td>
			<td>M-1700C-LB</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube magnetic separator rack</td>
			<td>Sergi Lab Supplies</td>
			<td>1005</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tube</td>
			<td>Corning&#160;</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube magnetic separator rack</td>
			<td>Sergi Lab Supplies</td>
			<td>1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7074P2</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop incubated shaker</td>
			<td>Bioer</td>
			<td>DIS-87999-3367802</td>
			<td>Bioer Thermocell Mixing Block MB-101</td>
		</tr>
		<tr>
			<td>CD9 (D3H4P) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>13403S</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroacetamide</td>
			<td>Sigma -Aldrich</td>
			<td>C0267-100G</td>
			<td>Used for alkylation of reduced sulfide groups. Freshly prepare 400 mM in water as stock solution.</td>
		</tr>
		<tr>
			<td>Ethyl acetate&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>E145-4</td>
			<td>Precipitates detergents</td>
		</tr>
		<tr>
			<td>Evosep One&#160;</td>
			<td>Evosep</td>
			<td></td>
			<td>Liquid chromatography system</td>
		</tr>
		<tr>
			<td>Evotips</td>
			<td>Evosep</td>
			<td>EV2013</td>
			<td>Sample loading for Evosep One system&#160;</td>
		</tr>
		<tr>
			<td>EVtrap</td>
			<td>Tymora Analytical</td>
			<td></td>
			<td>Functionalized magnetic beads, loading buffer, and washing buffer&#160;</td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF Membrane</td>
			<td>Sigma -Aldrich</td>
			<td>IPFL00010</td>
			<td>Blotting membrane&#160;</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Gel</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>Invitrogen NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4X)</td>
			<td>Invitrogen</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsep C18 15 x 75 x 1.9</td>
			<td>Bruker&#160;</td>
			<td>1893473</td>
			<td>Separation column&#160;</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail 2</td>
			<td>Sigma -Aldrich</td>
			<td>P5726-5ML</td>
			<td>100X, Phosphotase inhibitor.</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail 3</td>
			<td>Sigma -Aldrich</td>
			<td>P0044-1ML</td>
			<td>100X,&#160; Phosphotase inhibitor.&#160;</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce ECL Western Blotting Substrate</td>
			<td>ThermoFisher</td>
			<td>32106</td>
			<td>HRP substrate&#160;</td>
		</tr>
		<tr>
			<td>PolyMAC phosphopeptide enrichment kit</td>
			<td>Tymora Analytical</td>
			<td></td>
			<td>Polymer-based metal ion affinity capture (PolyMAC) for phosphopeptide enrichment</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate&#160;</td>
			<td>Sigma -Aldrich</td>
			<td>D6750-10G</td>
			<td>Detergent for lysis buffer. Prepare 120 mM in water as stock solution.</td>
		</tr>
		<tr>
			<td>Sodium lauroyl sarcosinate&#160;</td>
			<td>Sigma -Aldrich</td>
			<td>L9150-50G</td>
			<td>Detergent for lysis buffer. Prepare 120 mM in water as stock solution.</td>
		</tr>
		<tr>
			<td>timsTOF HT</td>
			<td>Bruker</td>
			<td></td>
			<td>Trapped ion-mobility time-of-flight mass spectrometry</td>
		</tr>
		<tr>
			<td>TopTip C-18 (10-200 &#956;L) tips&#160;</td>
			<td>Glygen</td>
			<td>TT2C18.96</td>
			<td>Desalting method</td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma -Aldrich</td>
			<td>471283-100ML</td>
			<td>For EV elution.&#160;</td>
		</tr>
		<tr>
			<td>Triethylammonium bicabonate buffer</td>
			<td>Sigma -Aldrich</td>
			<td>T7408-100ML</td>
			<td>1 M</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma -Aldrich</td>
			<td>302031-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Sigma -Aldrich</td>
			<td>C4706</td>
			<td>Used for reducion of disulfide bonds. Prepare 200 mM in water as stock solution. Aliquot the stock solution into small volume and store it in at-20&#176;C (avoid multiple freeze-thaw cycles).</td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C MIX</td>
			<td>ThermoFisher</td>
			<td>PIA41007</td>
			<td></td>
		</tr>
	</tbody>
</table>

chemical affinity-based isolation of extracellular vesicles from biofluids for proteomics and phosphoproteomics analysis

来自生物流体的细胞外囊泡 （EV） 最近在液体活检领域引起了极大的关注。它们由几乎所有类型的细胞释放，提供宿主细胞的实时快照，并包含丰富的分子信息，包括蛋白质，特别是那些具有翻译后修饰 （PTM） 的蛋白质，如磷酸化，作为细胞功能和疾病发生和进展的主要参与者。然而，由于当前 EV 分离方法的产量和杂质低，从生物流体中分离 EV 仍然具有挑战性，这使得 EV 货物（如 EV 磷蛋白）的下游分析变得困难。在这里，我们描述了一种基于功能化磁珠的快速有效的 EV 分离方法，用于从生物流体（如人尿液）中分离 EV 和 EV 分离后的下游蛋白质组学和磷酸化蛋白质组学分析。该方案实现了尿 EV 的高回收率以及 EV 蛋白质组和磷酸化蛋白质组的敏感谱。此外，本文还讨论了该协议的多功能性和相关技术考虑因素。

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.

该协议展示了从EV分离到下游蛋白质组学和磷酸化蛋白质组学分析的综合工作流程（图1）。对一式三份的尿液样本进行EV分离。分离的 EV 通过蛋白质印迹进行表征，随后进行基于质谱的蛋白质组学样品制备，包括蛋白质提取、酶消化和肽纯化。对于磷酸化蛋白质组学分析，基于金属离子功能化的可溶性纳米聚合物进一步富集了磷酸肽。多肽和磷酸肽样品均在数据无关模式下?...

Watch this Scientific Journal Video about Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery at JoVE.com

本方案为利用功能化磁珠有效分离尿细胞外囊泡提供了详细的描述。此外，它还包括后续分析，包括蛋白质印迹、蛋白质组学和磷酸化蛋白质组学。

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

我们正致力于基于细胞外囊泡的生物标志物发现。一个重要的步骤是有效和有选择地分离它们。EVtrap 基于磁珠分离，通过提供高通量、易于使用，特别是易于临床应用，真正可以帮助我们。我们一直在使用该协议来发现不同疾病（如乳腺癌，肾癌和帕金森病）的蛋白质和磷蛋白生物标志物。该生物标志物来源于生物流体。EV可能用于液体活检，这可以进一步改善临床诊断和治疗。目前，最常用的细胞外囊泡分离方法是超速离心和沉淀，但这些方法仅限于污染物的共分离，而且效率低。因此，我们的 EVtrap 方法将为分离 EV 提供更有效的方法，这将使方法更具可重复性和灵敏度，并且也有利于下游分析。我们正在做两件事。首先，我们正在继续开发EV隔离和下游分析的方法和协议，特别是基于大规模备份的分析。其次，由于EVtrap的优势，我们正在探索在临床领域的不同应用。

chemical-affinity-based-isolation-extracellular-vesicles-from

Research

JoVE Journal

Biochemistry

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

2.1K 次观看.  Purdue University. 我们正致力于基于细胞外囊泡的生物标志物发现。一个重要的步骤是有效和有选择地分离它们。EVtrap 基于磁珠分离，通过提供高通量、易于使用，特别是易于临床应用，真正可以帮助我们。我们一直在使用该协议来发现不同疾病（如乳腺癌，肾癌和帕金森病）的蛋白质和磷蛋白生物标志物。该生物标志物来源于生物流体。EV可能用于液体活检，这可以进一步改善临床诊?...

视频: 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 

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.

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.

科研

生物化学

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

To begin, collect the urine samples from healthy individuals. Centrifuge 12 milliliters of sample in a 15 milliliter conical tube to eliminate cell debris in large apoptotic bodies. Then, transfer 10 milliliters of the supernatant into a fresh 15 milliliter tube.Add one milliliter of loading buffer and 200 microliters of EV trap bead slurry to the sample. Incubate the sample at room temperature for 30 minutes with end over end rotation. To pellet the sample, place the 15 milliliter conical tube on a magnetic separator rack.Carefully remove the supernatant and re-suspend the beads in one milliliter of washing buffer. Then, transfer the suspension to a 1.5 milliliter micro centrifuge tube and gently pipette to re-suspend the extracellular vesicle or EV-bound beads. Place the micro centrifuge tube on a magnetic separator rack for 1.5 milliliter tubes.Using a P200 pipette, carefully aspirate the supernatant. Now, wash the beads with one milliliter of washing buffer, followed by two washes with one milliliter of PBS at room temperature. Incubate the beads with 100 microliters of freshly prepared 100 millimolar Triethanolamine for five minutes.Using 1.5 milliliter micro centrifuge tube magnetic separator rack, collect the eluded solution containing EVs. After combining the eluded solutions, dry the eluit, using a vacuum centrifuge concentrator at four degrees Celsius.

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

To begin, take the dried extracellular vesicle sample obtained from the human urine using the EV trap approach. Prepare a fresh lysis buffer with the shown components. Then add 100 microliters of lysis buffer to solubilize the dried EV sample.Heat the sample at 95 degrees Celsius for 10 minutes while shaking at 1, 100 RPM. After cooling the sample to room temperature, dilute it fivefold by adding 400 microliters of 50 millimolar TEAB. Measure the protein concentration using a BCA assay kit according to the manufacturer's instructions.Add trypsin and lysine C mix to the sample an incubate at 37 degrees celsius overnight with shaking at 1, 100 RPM. Then add 50 microliters of 10%trifluoroacetic acid or TFA to acidify the sample. Further, add 600 microliters of ethyl acetate to the samples and vortex the mixture for two minutes.Centrifuge the sample for three minutes at 20, 000 G and carefully remove the upper layer without disturbing the interface. Dry the aqueous phase using a vacuum centrifuge concentrator. Now resuspend the dried sample in 200 microliters of 0.1%TFA to acidify peptides.To desalt the sample, use a CA18 desalting tip and condition it with 200 microliters of 0.1%TFA and 80%acetonitrile, followed by two washes with 200 microliters of 0.1%TFA. Load the acidified peptide sample into the tip and wash it three times with 200 microliters of 0.1%TFA. Elute the peptides with 200 microliters of 0.1%TFA and 80%acetonitrile.After drawing the eluent as demonstrated, resuspend the dried to phosphoproteomics sample in 200 microliters of loading buffer for phosphopeptide enrichment. Add 50 microliters of the beads to the sample and vigorously shake for 20 minutes at room temperature. Load the sample with the beads into the fritted tip and centrifuge for one minute at 100 G.Wash the tip successively with 200 microliters of loading buffer, washing buffers one and two.Place the tip with beads into a new tube to collect the eluded phosphopeptides. Add 50 microliters of elution buffer to the tip and centrifuge for two minutes at 20 G.Then dry the eluded phosphopeptides using a vacuum centrifuge concentrator.

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

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

To begin, prepare proteome and phosphoproteome samples of the extracellular vesicles isolated from the urine samples using the EVtrap approach. Inject the samples into the trapped ion-mobility time-of-flight mass spectrometer through the liquid chromatography system. Separate the peptides into a 15-centimeter C18 column.For proteomics analysis, acquire data using the dia-PASEF method with a mass range per ramp to span from 300 to 1200 mass-to-charge ratio and ion-mobility constant from 0.6 to 1.50 1/Knot. For phosphoproteomics analysis, acquire data using a dia-PASEF acquisition method. Set the mass range per ramp to span from 400 to 1550 mass-to-charge ratio in ion-mobility constant from 0.6 to 1.50 1/Knot.Load the raw files into proteomic software. Set up the search parameters in the homo sapiens database. For Digest Type, select Specific, and under Enzymes, select TrypsinP.Define the Peptide Length to a minimum of 7 and a maximum of 52 and allow two missed cleavages. Then set the Maximum Variable Modifications to 5. The Fixed Modification should be Carbamidomethyl at Cystine, while the Variable Modifications should include Acetyl Protein N-Terminal, Oxidation at Methionine, and Phosphorylation at Serine, Threonine, and Tyrosine.Finally, set the false discovery rate for peptide spectrum match, peptide, and protein group to 0.01. In the LCMS and MS proteomic profiling, 2%of each sample revealed over 11, 000 unique peptides from approximately 2, 200 proteins. Notably, 72%of unique proteins were consistently found in all three replicates and 90 top EV markers and proteins were identified compared to the ExoCarta database.Quantitative precision was confirmed with a low-medium coefficient of variation of 5.7%signifying high reproducibility and reliability. For phosphoproteomics, 98%of each peptide sample yielded 800 unique phosphopeptides and 350 phosphoproteins. The enrichment resulted in 72%phosphoserine, 22%phosphothreonine, and 6%phosphotyrosine peptides.42%of phosphopeptides were identified in all replicates in a medium coefficient of variation of 21.8%was observed, indicating acceptable quantitative reproducibility.