Intact protein profiling of Chlorobium tepidum by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry.

Capillary isoelectric focusing (CIEF) coupled with reversed-phase liquid chromatography (RPLC) and electrospray ionization (ESI) mass spectrometry (MS) is shown to provide a liquid-based alternative to 2D-PAGE for intact protein profiling. This combination exhibits high resolution, sensitivity and throughput for protein profiling based on pI vs MW. The CIEF-RPLC-MS system described here facilitates the use of IEF markers for internal calibration of pI. It also provides a high dynamic range as evidenced by the detection of 100 pg (3 fmol) of a test protein spiked into 1 microg of a complex protein mixture. About 1200 individual proteins/polypeptides were detected from lysates of the green sulfur bacterium Chlorobium tepidum in a single <8 h run. The pI vs MW profile obtained from CIEF-RPLC-MS compares favorably with theoretical data derived from the C. tepidum genome and experimental data obtained from 2D-PAGE.