Mass Spectrometric Analysis of Synapsins in <i>Drosophila melanogaster</i> and Identification of Novel Phosphorylation Sites

Synapsins are synaptic vesicle-associated phosphoproteins that play a major role in the fine regulation of neurotransmitter release. In <i>Drosophila</i>, synapsins are required for complex behavior including learning and memory. Synapsin isoforms were immunoprecipitated from homogenates of wild-type <i>Drosophila</i> heads using monoclonal antibody 3C11. Synapsin null mutants (<i>Syn⁹⁷</i>) served as negative controls. The eluted proteins were separated by SDS-PAGE and visualized by silver staining. Gel pieces picked from five bands specific for wild type were analyzed by nano-LC−ESI−MS/MS following multienzyme digestion (trypsin, chymotrypsin, AspN, subtilisin, pepsin, and proteinase K). The protein was unambiguously identified with high sequence coverage (90.83%). A number of sequence conflicts were observed and the N-terminal amino acid was identified as methionine rather than leucine expected from the cDNA sequence. Several peptides from the larger isoform demonstrated that the in-frame UAG stop codon at position 582 which separates two large open reading frames is read through by tRNAs for lysine. Seven novel phosphorylation sites in <i>Drosophila</i> synapsin were identified at Thr-86, Ser-87, Ser-464, Thr-466, Ser-538, Ser-961, and Tyr-982 and verified by phosphatase treatment. No phosphorylation was observed at the conserved PKA/CaM kinase-I/IV site (RRFS, edited to RGFS) in domain A or a potential PKA site near domain E