Capillary isoelectric focusing electrospray ionization time-of-flight massspectrometry for protein analysis

On-line capillary isoelectric focusing (CIEF)-electrospray ionization time- of-flight mass spectrometry (ESI-TOFMS) as a two-dimensional separation sys tem is employed for high-resolution analysis of model proteins (myoglobin a nd beta-lactoglobulin) and human hemoglobin variants C, S, F, and A. The fo cused proteins in a polyacrylamide-coated capillary are mobilized by replac ing the sodium hydroxide catholyte with a sheath liquid solution containing methanol-water-acetic acid in a volume ratio of 80:19:1. The use of sheath liquid also establishes the electrical connection at the CIEF capillary te rminus, which serves to define the electric field along the CLEF capillary and apply an electric voltage for electrospray ionization. At the end of th e CIEF capillary, the mobilized protein zones are analyzed and directly ide ntified by ESI-TOFMS. The TOFMS accumulates spectra at 5000 Hz frequency an d one averaged spectrum is acquired per second during the CIEF-ESI-TOFMS me asurements. (C) 1999 John Wiley & Sons, Inc.