MATRIX-ASSISTED LASER-DESORPTION MASS-SPECTROMETRIC PEPTIDE-MAPPING OF PROTEINS SEPARATED BY 2-DIMENSIONAL GEL-ELECTROPHORESIS - DETERMINATION OF PHOSPHORYLATION IN SYNAPSIN-I

A technique is described for the rapid, sensitive analysis of posttran slational modifications of proteins that have been separated by 2-dime nsional electrophoresis and blotted onto a membrane with a cationic su rface. The isolated protein spots visualized by reverse staining of th e blotting membrane are excised, washed, and subjected to chemical (cy anogen bromide) and/or enzymatic (endoproteinase Lys-C) degradation di rectly on the membrane. The resulting mixture of peptide fragments is extracted from the membrane into a solution that is compatible with ma trix-assisted laser desorption mass spectrometric analysis and analyze d without fractionation. Relatively accurate (+/- 1 Da) mass determina tion of these peptide fragments provides a facile and sensitive means for detecting the presence of modifications and for correlating such m odifications with the differential mobility of different isoforms of a given protein during 2-dimensional electrophoresis. The technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib, neuronal phosphoproteins that are believed to function in t he regulation of neurotransmitter release and are substrates for cAMP and Ca2+/calmodulin-dependent protein kinases, which appear to control their biological activity.