Mass spectrometric analysis of synapsins in Drosophila melanogaster and identification of novel phosphorylation sites

Tulip Nuwal, Seok Heo, Gert Lubec, Erich Buchner

Research output: Contribution to journalArticlepeer-review

Abstract

Synapsins are synaptic vesicle-associated phosphoproteins that play a major role in the fine regulation of neurotransmitter release. In Drosophila, synapsins are required for complex behavior including learning and memory. Synapsin isoforms were immunoprecipitated from homogenates of wild-type Drosophila heads using monoclonal antibody 3C11. Synapsin null mutants (Syn 97) 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 Drosophila 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.

Original languageEnglish (US)
Pages (from-to)541-550
Number of pages10
JournalJournal of proteome research
Volume10
Issue number2
DOIs
StatePublished - Feb 4 2011
Externally publishedYes

Keywords

  • Drosophila melanogaster
  • Immunoprecipitation
  • Mass spectrometry
  • Phosphorylation
  • Post-translational modification
  • Synapsin
  • UAG suppression

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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