Combination of capillary isoelectric focusing and liquid chromatography with electrospray ionization mass spectrometry for protein characterization

Combination of capillary isoelectric focusing (CIEF) and liquid chromatography (LC) with electrospray ionization mass spectrometry (ESIMS) is utilized for protein characterization;Both CIEF-ESIMS and LC-MS are studied for phosphoprotein characterization. At the intact protein level, on-line CTEF-ESIMS is applied for phosphorylation analysis to obtain the two dimensional information: isoelectric point and molecular mass. At the peptide level, on-line LC-ESI-MS and LC-ESI neutral loss MS/MS were highly specific for the determination of sites of phosphorylation and successfully applied on the tryptic digest of tau proteins phosphorylated by different kinases;On-line combination of CIEF with ESI-TOF MS is demonstrated for high resolution analysis of some model proteins and human hemoglobin variants. The successful coupling of these two techniques optimizes the efficiency of CIEF separation since TOF MS is able to produce a full spectrum in the shortest response time among all kinds of mass analyzers