Two-dimensional separation system by on-line hyphenation of capillary isoelectric focusing with pressurized capillary electrochromatography for peptide and protein mapping

A novel on-line 2-D system was developed for peptide and protein mapping. The system combines capillary IEF (cIEF) with pressurized CEC (pCEC) using a micro-injection valve as the interface. Sample fractions, which were focused and separated in the first-dimension cIEF based on their differences in pIs, were electrically mobilized and further successively resolved by their differences in size, hydrophobicity, and electrophoretic mobility in the second-dimension pCEC. In the presented system, the valve interface was free of the external electric field in two dimensions for the purpose of stabilization, safety, and facilitating manipulation. In the first dimension, cIEF separation was executed by a one-step method to simplify the operation procedure. Moreover, a home-made electrical decoupler was introduced to isolate the micro-injection valve from the cIEF electric field. For the second dimension, taking advantage of the combination of hydrodynamic flow with EOF, reversed-phase pCEC not only offers on-column refocusing the effluent fractions, but also brings enhanced separation resolution and elution speed. Separation effectiveness of this 2-D system was demonstrated by the analysis of tryptic digest of BSA and human red blood cell lysate. A theoretical peak capacity of approximately 24,000 has been achieved for BSA digest, which proves its promising potential for the application in proteomics.