Construction of discontinuous capillary isoelectric focusing system and its application in pre-fractionation of exosomal proteins

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 (R 2  = 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.