CAPILLARY COLUMN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ELECTROSPRAY-IONIZATION TRIPLE-STAGE QUADRUPOLE MASS-SPECTROMETRIC ANALYSIS OF PROTEINS SEPARATED BY 2-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS - APPLICATION TO CEREBELLAR PROTEIN MAPPING

A method is presented for the structural characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PA GE). The method includes separation of a protein mixture by 2D-PAGE, r ecovery of proteins from the gel spots revealed by copper staining and analysis of the proteins by triple-stage quadrupole mass spectrometry using an electrospray ionization interface (ESI-TSQMS). Prior to the mass spectrometric analysis, the extracted proteins were passed throug h a small reversed-phase column (10 x 4.0 mm I.D.) to remove salts and gel-derived contaminants and then introduced into the mass spectromet er through a reversed-phase capillary column with 0.25 mm LD. Applicat ion of the method to the analysis of rat cerebellar proteins suggests that the molecular mass could be accurately determined with sub-picomo le amounts of protein samples derived from one or two 2D gels. The met hod was also useful for peptide mapping and determination of amino aci d sequences of proteins micro-prepared from the 2D gel. Because 2D-PAG E has an excellent resolving power in protein separation and because c apillary LC-ESI-TSQMS provides structural information with very small amounts of samples, the combined system of 2D-PAGE and capillary LC-ES I-TSQMS described here should allow wide applications to molecular stu dies of genes and proteins, such as identifications of protein spots o n 2D gels, confirmation of gene/protein sequences and analysis of post -translational modification of proteins present naturally in tissue/ce ll extracts or expressed by recombinant DNA techniques.