Proteome of DiFi and CC-CR supermeres purified by FPLC with or without additional affinity purification for TGFBi

The cell seretome is diverse and contains extracellular secreted vesicles (EVs) as well as non-vesicular extracellular nanoparticles (NVEPs) called exomeres and supermeres, all of which carry different kinds of cell signaling cargos. One supermere enriched cargo is transforming growth factor beta induced (TGFBi) protein. We have created TGFBi-neon green-His tagged expressing cell lines in the microsatellite stable (MSS) colorectal cancer cell line DiFi and another one in CC-CR cells with microsatellite instability (MSI). We have developed a fast protein liquid chromatography (FPLC) method for isolating EVs, exomeres and supermeres from biofluids. We used either FPLC or FPLC and then nickel columns that bind His tags to isolate supermeres from DiFi or CC-CR cells. We utilized a proteomics approach acquiring LC-MS/MS data to compare FPLC to FPLC and His tagged TGFBi-containing purified supermeres from DiFi and CC-CR cells. This was done using hollow fiber 3D bioreactor production of conditioned media from these cell lines.

细胞分泌组（cell secretome）种类多样，包含细胞外分泌囊泡（extracellular secreted vesicles, EVs）以及被称为外泌颗粒（exomeres）和超微颗粒（supermeres）的非囊泡型细胞外纳米颗粒（non-vesicular extracellular nanoparticles, NVEPs），所有上述组分均携带不同类型的细胞信号载荷。其中一种富集于超微颗粒的载荷为转化生长因子β诱导蛋白（transforming growth factor beta induced, TGFBI）。我们已在微卫星稳定（microsatellite stable, MSS）结直肠癌细胞系DiFi中构建了携带TGFBi-荧光绿-His标签的融合表达细胞系，并在具有微卫星不稳定性（microsatellite instability, MSI）的CC-CR细胞中完成了另一组细胞系构建。我们开发了一种快速蛋白液相色谱（fast protein liquid chromatography, FPLC）方法，可从生物流体中分离细胞外分泌囊泡、外泌颗粒与超微颗粒。我们分别采用两种策略从DiFi或CC-CR细胞中分离超微颗粒：其一为单独使用快速蛋白液相色谱法，其二为先通过快速蛋白液相色谱，再辅以可结合His标签（His tags）的镍柱。我们借助蛋白质组学方法获取液相色谱-串联质谱（LC-MS/MS）数据，以对比分析从两种细胞系中分离得到的、分别经快速蛋白液相色谱法纯化，以及经His标签亲和纯化且携带TGFBI的超微颗粒。本研究通过中空纤维三维生物反应器（hollow fiber 3D bioreactor）制备上述细胞系的条件培养基，以此完成全部实验流程。