Capillary isoelectric focusing-mass spectrometry: Coupling strategies and applications

IEF on immobilized pH gradient strips is a widespread tool for protein separation, especially as first dimension in commonly utilized 2DE. In the latter arrangement, separations are based on two orthogonal molecular characteristics according to pI in the first and molecular weight in the second dimension. However, the approach is time consuming, quantification is difficult and MS can be applied only offline. Capillary IEF and related IEF techniques in combination with MS provide similar information. The major benefits are high mass resolution and mass accuracy, reproducibility, speed, automation, and quantification by using a high-resolution mass spectrometer. However, online hyphenation of CIEF with MS is interfered by the ampholytes, acids, and bases needed for high-resolution IEF. This review will give an overview about important coupling techniques, like low ampholyte concentration, interim separation by chromatography, or the use of a dialysis interface to separate the analytes from interfering substances. It is focused on strategies which allow sensitive MS detection of CIEF-separated analytes. In addition, proteomic and biopharmaceutical applications of capillary IEF techniques combined with MS are briefly summarized.