Capillary isoelectric focusing – mass spectrometry for a shotgun approach in proteomics

Abstract

Proteomics is the systematic analysis of proteins and their expression patterns in biological systems. It involves the localization, isolation, identification and functional characterization of the proteins in an organism. One particular technique used in these studies is the shotgun approach. This involves enzymatic digestion of the proteins, followed by separation and analysis by tandem mass spectrometry (MS/MS).In this research, the application of capillary isoelectric focusing (CIEF) for the shotgun approach is studied. During CIEF-analysis, amphoteric compounds are separated according to their pI-values, in a pH gradient formed under the influence of an electric field. Normally, carrier ampholytes are used to establish a linear pH gradient. Because of their involatile nature, they cause dramatic suppression of MS signal intensity of the analyte ions.In this research, CIEF analysis of digested proteins is based on autofocusing, using only limited amounts of carrier ampholytes as spacers. The efficacy and the limitations of this approach are studied. Complex protein mixtures are successfully analyzed by CIEF-MS/MS.Furthermore, proteolytic 18O labeling is successfully applied in this approach for differential analysis. Overall, CIEF-MS provides a valuable alternative to LC-MS analysis of complex peptide mixtures, both in a qualitative way as well as in a quantitative way.