Deconstruction of Heterogeneity of Size-Dependent Exosome Subpopulations from Human Urine by Profiling N‑Glycoproteomics and Phosphoproteomics Simultaneously

The heterogeneous populations of exosomes with distinct nanosize have impeded our understanding of their corresponding function as intercellular communication agents. Profiling signaling proteins packaged in each size-dependent subtype can disclose this heterogeneity of exosomes. Herein, new strategy was developed for deconstructing heterogeneity of distinct-size urine exosome subpopulations by profiling N-glycoproteomics and phosphoproteomics simultaneously. Two-dimension size exclusion liquid chromatography (SEC) was utilized to isolate large exosomes (L-Exo), medium exosomes (M-Exo), and small exosomes (S-Exo) from human urine samples. Then, hydrophilic carbonyl-functionalized magnetic zirconium-organic framework (CFMZOF) was developed as probe for capturing the two kinds of post-translational modification (PTM) peptides simultaneously. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with database search was used to characterize PTM protein contents. We identified 144 glycoproteins and 44 phosphoproteins from L-Exo, 156 glycoproteins, and 46 phosphoproteins from M-Exo and 134 glycoproteins and 10 phosphoproteins from S-Exo. The ratio of the proteins with simultaneous glycosylation and phosphorylation is 11%, 9%, and 3% in L-Exo, M-Exo, and S-Exo, respectively. Based on label-free quantification intensity results, both principal component analysis and Pearson’s correlation coefficients indicate that distinct-size exosome subpopulations exist significant differences in PTM protein contents. Analysis of high abundance PTM proteins in each exosome subset reveals that the preferentially packaged PTM proteins in L-Exo, M-Exo, and S-Exo are associated with immune response, biological metabolism, and molecule transport processes, respectively. Our PTM proteomics study based on size-dependent exosome subtypes opens a new avenue for deconstructing the heterogeneity of exosomes.具有不同纳米尺寸的异质性外泌体（exosomes）群体，长期阻碍了我们对其作为细胞间通讯介质对应功能的认知。解析每种尺寸依赖性亚型所包裹的信号蛋白特征，可揭示外泌体的这种异质性。 本研究开发了一种新策略，通过同时开展N-糖基化蛋白质组学（N-glycoproteomics）与磷酸化蛋白质组学（phosphoproteomics）分析，解析人类尿液中不同尺寸外泌体亚群的异质性。 采用二维尺寸排阻液相色谱（size exclusion liquid chromatography, SEC）从人类尿液样本中分离得到大体积外泌体（L-Exo）、中体积外泌体（M-Exo）与小体积外泌体（S-Exo）。 随后，本研究开发了亲水羰基功能化磁性锆有机框架（hydrophilic carbonyl-functionalized magnetic zirconium-organic framework, CFMZOF）作为探针，可同时捕获两类翻译后修饰（post-translational modification, PTM）肽段。 最后，结合液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）与数据库检索技术，对翻译后修饰蛋白含量进行表征。 本研究从L-Exo中鉴定得到144个糖蛋白与44个磷酸蛋白，从M-Exo中鉴定得到156个糖蛋白与46个磷酸蛋白，从S-Exo中鉴定得到134个糖蛋白与10个磷酸蛋白。L-Exo、M-Exo与S-Exo中同时携带糖基化与磷酸化修饰的蛋白占比分别为11%、9%与3%。 基于无标记定量（label-free quantification）强度结果，主成分分析（principal component analysis）与皮尔逊相关系数（Pearson’s correlation coefficients）分析均表明，不同尺寸的外泌体亚群在翻译后修饰蛋白含量上存在显著差异。 对各外泌体亚群中的高丰度翻译后修饰蛋白进行分析后发现，L-Exo、M-Exo与S-Exo中优先包裹的翻译后修饰蛋白分别与免疫应答、生物代谢及分子转运过程相关。 本项基于尺寸依赖性外泌体亚型的翻译后修饰蛋白质组学研究，为解析外泌体异质性开辟了新途径。