Exosomal proteins play an important role in screening disease biomarker and clinic diagnosis. However, low concentration, complex composition and difficulty in extraction hindered the study of their structure and properties. In this study, a new protein pre-fractionation system, discontinuous monolithic immobilized pH gradient capillary isoelectric focusing (DM-IPG-CIEF), was constructed by reassembling pieces of monolithic IPG-CIEF column to create small cavity with well-defined pH range. The monolithic IPG-CIEF column exhibited good linearity (R² = 0.9997) and preparation and operation reproducibility. Then removal of high-abundant proteins and enrichment of low-abundant proteins were performed by the DM-IPG-CIEF system successfully. The system was applied to pre-fractionation and enrichment of exosomal proteins extracted from HeLa cells. The exosomal proteins with pI in the range of 6.0–9.0 were focused in enrichment cavity and collected. According to nano-HPLC-MS/MS analysis, 130 proteins in this range were identified, indicating great potential of the DM-IPG-CIEF system in pretreatment of exosomal proteins.

外泌体蛋白在筛选疾病生物标志物和临床诊断中起着重要作用。然而，低浓度，复杂的组成和提取困难阻碍了对其结构和性质的研究。在这项研究中，通过重新组装整体式IPG-CIEF色谱柱以创建具有明确pH范围的小腔，构建了一种新的蛋白质预分离系统，即不连续整体式固定pH梯度毛细管等电聚焦（DM-IPG-CIEF）。整体式IPG-CIEF色谱柱表现出良好的线性（R ² = 0.9997）以及准备和操作的可重复性。然后通过DM-IPG-CIEF系统成功地去除了高丰度蛋白并富集了低丰度蛋白。该系统用于从HeLa细胞提取的外泌体蛋白的预分级分离和富集。pI在6.0-9.0范围内的外泌体蛋白集中在富集腔中并收集。根据纳米HPLC-MS / MS分析，鉴定出130种蛋白质，表明DM-IPG-CIEF系统在外泌体蛋白质预处理中具有巨大潜力。