NOVEL TECHNIQUES FOR IDENTIFICATION AND CHARACTERIZATION OF PROTEINS LOADED ON GELS IN FEMTOMOLE AMOUNTS

A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-des orption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix, There, they are digested i n the presence of (H2O)-O-18 and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (P SD) spectra with theoretical spectra of candidate isobaric peptides us ing a computer algorithm called MassFrag, and (3) by a manual readout of the O-18/O-16-labeled fragmentation ions in the PSD spectra.