Separation of tryptic digests using a modified buffer in pressurized capillary. Electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer.

A rapid and efficient separation method using pressurized capillary electrochromatography (pCEC) has been developed to separate protein digests. The effects of mobile-phase ion conductivity, pH, and column size on separation speed and column efficiency were studied and optimized. The pCEC method was demonstrated to provide enhanced speed with more efficient and selective separation than HPLC by performing separations of a bovine beta-lactoglobulin A digest. A human hemoglobin digest was separated by pCEC under optimized conditions, and more than 20 peaks were separated in less than 20 min. Using an ion trap storage/reflectron time-of-flight mass spectrometer, coeluting components were clearly identified, and the resolving power of the pCEC method was further enhanced.