Hypersensitive Measurement of Proteins by Capillary Isoelectric Focusing and Liquid Chromatography-Mass Spectrometry

Hypersensitive Measurement of Proteins by Capillary Isoelectric Focusing and Liquid Chromatography-Mass Spectrometry 

Department of Chemistry and Biochemistry, University of Delaware. Newark, DE 19716, USA 

Chromalographic Methods in Clinical Chemistry and Toxicology Edited by R. L. Bertholf and R. E. Winecker 2007 John Wiley Sons, Ltd 

Unfortunately, substantial problems remain with the CIEF-MS approach. Most importantly, the ampholyte used in CIEF co-elutes with the analyte and perturbs the electrospray ionization (ESI) process in a manner that suppresses analyte signal intensity and degrades mass resolution. In addition, the resolution of CIEF alone is not sufficient to separate proteins completely in complex samples. When proteins co-elute from an ESI source, some 

In our work, an improved CIEF-RPLC-MS system has been developed for protein separation and characterization. A microdialysis membrane separates the cathodic cell from the capillary column. During the focusing step, catholyte is able to traverse the membrane but protein molecules cannot. After focusing, proteins are hydrodynamically pushed past the membrane to a microselection valve that collects and transfers fractions to the RPLC system for further separation. This configuration permits a linear pH gradient to be maintained in the separation capillary with no voltage applied to the microselsection valve. The cathodic cell and microselection valve can easily and safely be retrofitted into commercial instruments. 

---