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Proteomic analysis of synaptosomes using isotope‐coded affinity tags and mass spectrometry
Author(s) -
Schrimpf Sabine P.,
Meskenaite Virginia,
Brunner Erich,
Rutishauser Dorothea,
Walther Pascal,
Eng Jimmy,
Aebersold Ruedi,
Sonderegger Peter
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401198
Subject(s) - synaptic vesicle , postsynaptic density , postsynaptic potential , chemistry , proteome , synaptosome , biochemistry , snap25 , synaptic vesicle recycling , vesicle , receptor , membrane
Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope‐coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine‐containing peptides were isolated by affinity chromatography. The peptides were identified by microcapillary liquid chromatography‐electrospray ionisation MS/MS (μLC‐ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane‐bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.