Construction Of An Interstitial Capillary Isoelectric Focusing System For Pre-fractionnation And Its Application In Proteomics

Proteomics is characterized by complex structure, variety and wide dynamic range, etc. Protein pre-fractionation can reduce the complexity of samples and contribute to the establishment of systems with high sensitivity and high efficiency. An interstitial capillary isoelectric focusing system for pre-fractionation was constructed and used for pre-fractionation of protein samples.To establish the pre-fractionation system, a monolithic immobilized pH gradient capillary isoelectric focusing (M-IPG-CIEF) column was prepared by’in situ polymerization focusing method’using hydrophilic acrylamide monomer. Separation capability, linearity and corresponding affecting factors were investigated by the separation of a four-protein mixture. The result showed that linear relationship between elution time and pi values was satisfactory with correlation of R2>0.997at the flow rate of10μL/min. An interstitial capillary isoelectric focusing system for pre-fractionnation was constructed by reconnecting pieces of M-IPG column with Teflon tube to create a small cavity between two pieces. Working principle, separation capability and linearity of the pre-fractionation system were illustrated through the separation of four standard proteins. And through the accurate control of pH range of each piece, bovine serum albumin (BSA) was focused in the cavity. The result showed that the interstitial capillary isoelectric focusing system kept good linearity and separation capability of M-IPG column.Removal of high-abundant proteins and enrichment of low-abundant proteins were accomplished on the interstitial capillary isoelectric focusing system for pre-fractionation. By focusing high concentration protein in separation cavity, albumin egg was easily removed with a clearance rate of77%. Moreover, bovine milk was selected as real sample to evaluate the capability of enriching low-abundance proteins or peptides in pI range from4.7to5.1on the interstitial capillary isoelectric focusing system. And a five times enhancement in peak area was observed. It was confirmed that the systempossessed good stability. In addition, pieces of the interstitial capillary isoelectric focusing system were connected with C8capillary column or CZE column coating with polyacrylamide (LPAA) through a Teflon tube seamless, and the two system were used to separate either bovine milk or its trypsin digestion products. Proteins/peptides couldn’t be separated on CIEF were separated. Both improvements in peak capacity and separation ability were obtained.