Mass spectrometic detection for capillary isoelectric focusing separationsof complex protein mixtures

Capillary isoelectric focusing (CIEF) can provide high-resolution separatio ns of complex protein mixtures, but until recently it has primarily been us ed with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein ch aracterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectro metry and its application to proteome characterization. Studies have reveal ed 400-1000 putative proteins in the mass range of 2-100 kDa from total inj ections of similar to 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for q uantitative proteome-wide measurements of protein expression. The ability t o make such comprehensive and precise measurements of differences in protei n expression in response to cellular perturbations should provide new insig hts into complex cellular processes.