Preparation of postsynaptic density fraction from hippocampal slices and proteomic analysis.
Hippocampal slices offer an excellent experimental system for the study of activity-induced changes in the postsynaptic density (PSD). While studies have documented electrophysiological and structural changes at synapses in response to precise manipulations of hippocampal slices, parallel biochemical and proteomic analyses were hampered by the lack of subcellular fractionation techniques applicable to starting tissue about three orders of magnitude smaller than that used in conventional protocols. Here, we describe a simple and convenient method for the preparation of PSD fractions from hippocampal slices and the identification of its components by proteomic techniques. The "micro PSD fraction" obtained following two consecutive extractions of a synaptosomal fraction with Triton X-100 shows a significant enrichment in the marker protein PSD-95. Thin section electron microscopy shows PSDs similar to those observed in situ. However, other particulate material, especially myelin, and membrane vesicles are also present. The composition of the PSD fraction from hippocampal slices was analyzed by 2D LC/MS/MS. The proteomic approach which utilizes as little as 10microg total protein allowed the identification of >100 proteins. Many of the proteins detected in the fraction are the same as those identified in conventional PSD preparations including specialized PSD-scaffolding proteins, signaling molecules, cytoskeletal elements as well as certain contaminants. The results show the feasibility of the preparation of a PSD fraction from hippocampal slices of reasonable purity and of sufficient yield for proteomic analyses. In addition, we show that further purification of PSDs is possible using magnetic beads coated with a PSD-95 antibody.
Dosemeci
Ayse
A
Laboratory of Neurobiology, NINDS, NIH, Bethesda, MD 20892, USA. dosemeca@mail.nih.gov
Tao-Cheng
Jung-Hwa
JH
Vinade
Lucia
L
Jaffe
Howard
H
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D000076122:Disks Large Homolog 4 Protein; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013569:Synapses / Q000378:metabolism*
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins