Development of capillary electrophoresis and mass spectrometry methods in protein analysis.

Abstract

Capillary electrophoresis combined with mass spectrometric analysis has been applied to protein analysis at low-femtomole levels. An on-line CE/MS system has been developed with the use of the ion trap/reflectron time-of-flight mass spectrometer (IT/reTOFMS) as the mass detector. This method provides rapid and sensitive detection for fast CE separations due to its high mass spectral acquisition speed. The CE separation has been performed using a polybrene capillary coating to control the electroosmotic flow. The CE/IT/reTOFMS was applied in the characterization of hemoglobin variants by analyzing their mass peptide profiles resulting from enzymatic digestion of the proteins. Comparison of the peptide mass maps and the mass differences can readily identify the hemoglobin mutations, and single amino acid mutations can be detected. In order to analyze proteins extracted from the cells, capillary isoelectric focusing separation was interfaced to matrix-assisted laser desorption/ionization (MALDI) MS for bacterial protein analysis. A nitrocellulose membrane was used for sample collection from capillary isoelectric focusing (CIEF) separation and as the MALDI MS analysis substrate. This method provides high speed and high mass accuracy compared with the traditional 2-Dimensional (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel electrophoresis method. Two-dimensional separation by coupling CIEF to capillary zone electrophoresis (CZE) was investigated. The transfer of sample between the two separation columns and exchange of separation buffers were accomplished by implementing a hollow fiber tube between the columns. Complex peptide and protein mixtures were separated by CIEF/CZE with UV or mass detection. This 2-D separation system shows great potential in analysis of complex mixture such as protein extracts from the cells with mass spectrometric detection.