IDENTIFICATION OF MOUSE-LIVER PROTEINS ON 2-DIMENSIONAL ELECTROPHORESIS GELS BY MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY OF IN-SITU ENZYMATIC DIGESTS

A number of proteins from a silver-stained two-dimensional (2-D) elect rophoresis gel of mouse liver whole-cell lysate were identified by pep tide mass mapping and sequence database searching. The excised protein spots were processed by in situ reduction and alkylation, followed by Lys-C digestion. The masses of the resulting peptide mixtures were me asured with a matrix-assisted laser desorption/ionization (MALDI) refl ectron-time-of-flight mass spectrometer. These masses were used succes sfully to search a protein sequence database. Optimized silver stainin g and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provi ded by delayed extraction were important for high specificity in the d atabase search. Fragment ion data obtained by MALDI post-source decay (PSD) measurements not only provided confirmation of peptide identific ation, but could be used to identify the protein from a single peptide without spectral interpretation.