Capillary isoelectric focusing/reversed phase liquid chromatography/mass spectrometry.

The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into a mass spectrometer for protein identification and quantification. An integrated protein separation platform, combining capillary isoelectric focusing (CIEF) with reversed phase liquid chromatography (RPLC), is described to provide high resolving power for the analysis of complex protein mixtures. Thus, the proteins are systematically resolved according to their differences in isoelectric point and hydrophobicity using combined CIEF/RPLC separations. A key feature of the CIEF-based multidimensional separation platform is the elimination of protein loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein profiles within small cell populations or limited tissue samples.