Exosomes are cell-derived functional microparticles which exist in most body fluids. They carry abundant signaling molecules to transfer information between cells and microenvironment. Research on exosomes’ heterogeneity and constitute variations has been a heated topic in recent years. In this work, size-dependent sub-proteome analysis of urinary exosomes was investigated by size exclusion chromatography (SEC) firstly. The particle size of urinary exosomes is distributed in four main ranges naturally. We found out that these fractions contained sub-proteomes with great difference in constitution. In each fraction, 206, 134, 157, and 276 unique proteins were identified by LC-MS/MS. Differential expression of exosomal markers such as TSG101, CD9, CD63, and caveolin-1 was observed in these fractions by western blots. Biological function annotation indicated that the proteins identified in each fraction were involved in different molecular and cellular processes. It is proven that SEC can serve as an efficient analytical tool for exosomes isolation and fractionation. This work provides a new strategy to classify exosomes into sub-populations for comprehensive study of heterogeneous functionalities.

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外来体是存在于大多数体液中的细胞来源的功能性微粒。它们携带丰富的信号分子在细胞和微环境之间传递信息。近年来，关于外泌体异质性和组成变异的研究一直是热门话题。在这项工作中，首先通过尺寸排阻色谱法（SEC）研究了尿液外泌体的尺寸依赖性亚蛋白质组分析。泌尿外泌体的粒径自然分布在四个主要范围内。我们发现这些级分包含的子蛋白质组在组成上有很大差异。在每个部分中，通过LC-MS / MS鉴定出206、134、157和276种独特的蛋白质。通过蛋白质印迹法在这些级分中观察到外泌体标记物（例如TSG101，CD9，CD63和小窝蛋白1）的差异表达。生物学功能注释表明，每个级分中鉴定出的蛋白质都参与了不同的分子和细胞过程。事实证明，SEC可作为外泌体分离和分离的有效分析工具。这项工作提供了一种新的策略，将外泌体分类为亚群，以全面研究异质功能。

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