A proteomic survey of rat cerebral cortical synaptosomes

Frank A. Witzmann, Randy J. Arnold, Fengju Bai, Petra Hrncirova, Mark W. Kimpel, Yehia S. Mechref, William J. McBride, Milos V. Novotny, Nathan M. Pedrick, Heather N. Ringham, Jay R. Simon

Research output: Contribution to journalReview article

77 Citations (Scopus)

Abstract

Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.

Original languageEnglish (US)
Pages (from-to)2177-2201
Number of pages25
JournalProteomics
Volume5
Issue number8
DOIs
StatePublished - May 1 2005

Fingerprint

Synaptosomes
Proteomics
Rats
Proteins
Syntaxin 1
Synapsins
GAP-43 Protein
Surveys and Questionnaires
Excitatory Amino Acid Transporter 2
Excitatory Amino Acid Transporter 1
Synaptotagmin II
Intracellular Signaling Peptides and Proteins
Synaptotagmin I
Vesicle-Associated Membrane Protein 2
Gels
Septins
Voltage-Dependent Anion Channels
GABA Plasma Membrane Transport Proteins
Synaptotagmins
Post-Synaptic Density

Keywords

  • Cerebral cortex
  • Mass spectrometry
  • Proteome
  • Rat
  • Synaptic proteins
  • Synaptosomes
  • Two-dimensional gel electrophoresis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Witzmann, F. A., Arnold, R. J., Bai, F., Hrncirova, P., Kimpel, M. W., Mechref, Y. S., ... Simon, J. R. (2005). A proteomic survey of rat cerebral cortical synaptosomes. Proteomics, 5(8), 2177-2201. https://doi.org/10.1002/pmic.200401102

A proteomic survey of rat cerebral cortical synaptosomes. / Witzmann, Frank A.; Arnold, Randy J.; Bai, Fengju; Hrncirova, Petra; Kimpel, Mark W.; Mechref, Yehia S.; McBride, William J.; Novotny, Milos V.; Pedrick, Nathan M.; Ringham, Heather N.; Simon, Jay R.

In: Proteomics, Vol. 5, No. 8, 01.05.2005, p. 2177-2201.

Research output: Contribution to journalReview article

Witzmann, FA, Arnold, RJ, Bai, F, Hrncirova, P, Kimpel, MW, Mechref, YS, McBride, WJ, Novotny, MV, Pedrick, NM, Ringham, HN & Simon, JR 2005, 'A proteomic survey of rat cerebral cortical synaptosomes', Proteomics, vol. 5, no. 8, pp. 2177-2201. https://doi.org/10.1002/pmic.200401102
Witzmann FA, Arnold RJ, Bai F, Hrncirova P, Kimpel MW, Mechref YS et al. A proteomic survey of rat cerebral cortical synaptosomes. Proteomics. 2005 May 1;5(8):2177-2201. https://doi.org/10.1002/pmic.200401102
Witzmann, Frank A. ; Arnold, Randy J. ; Bai, Fengju ; Hrncirova, Petra ; Kimpel, Mark W. ; Mechref, Yehia S. ; McBride, William J. ; Novotny, Milos V. ; Pedrick, Nathan M. ; Ringham, Heather N. ; Simon, Jay R. / A proteomic survey of rat cerebral cortical synaptosomes. In: Proteomics. 2005 ; Vol. 5, No. 8. pp. 2177-2201.
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