Proteomic analysis of synaptosomes using isotope-coded affinity tags and mass spectrometry.
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 (muLC-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.
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Sabine P
SP
Schrimpf
Virginia
V
Meskenaite
Erich
E
Brunner
Dorothea
D
Rutishauser
Pascal
P
Walther
Jimmy
J
Eng
Ruedi
R
Aebersold
Peter
P
Sonderegger
D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002247:Carbon Isotopes; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D051379:Mice; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics; D013574:Synaptosomes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D002247:Carbon Isotopes; D009419:Nerve Tissue Proteins