Quantitative Proteomics and Protein Network Analysis of Hippocampal Synapses of CaMKIIα Mutant Mice

Abstract

Quantitative analysis of synaptic proteomes from specific brain regions is important for our understanding of the molecular basis of neuroplasticity and brain disorders. In the present study we have optimized comparative synaptic proteome analysis to quantitate proteins of the synaptic membrane fraction isolated from the hippocampus of wild type mice and 3'UTR-calcium/calmodulin-dependent kinase II alpha mutant mice. Synaptic proteins were solubilized in 0.85% RapiGest and digested with trypsin without prior dilution of the detergent, and the peptides from two groups of wild type mice and two groups of CaMKIIalpha 3'UTR mutants were tagged with iTRAQ reagents 114, 115, 116, and 117, respectively. The experiment was repeated once with independent biological replicates. Peptides were fractionated with tandem liquid chromatography and collected off-line onto MALDI metal plates. The first iTRAQ experiment was analyzed on an ABI 4700 proteomics analyzer, and the second experiment was analyzed on an ABI 4800 proteomics analyzer. Using the criteria that the proteins should be matched with at least three peptides with the highest CI% of a peptide at least 95%, 623 and 259 proteins were quantified by a 4800 proteomics analyzer and a 4700 proteomics analyzer, respectively, from which 249 proteins overlapped in the two experiments. There was a 3 fold decrease of calcium/calmodulin-dependent kinase II alpha in the synaptic membrane fraction of the 3'UTR mutant mice. No other major changes were observed, suggesting that the synapse protein constituents of the mutant mice were not substantially altered. A first draft of a synaptic protein interaction network has been constructed using commercial available software, and the synaptic proteins were organized into 10 (interconnecting) functional groups belonging to the pre- and postsynaptic compartments, e.g., receptors and ion channels, scaffolding proteins, cytoskeletal proteins, signaling proteins, adhesion molecules, and proteins of synaptic vesicles and those involved in membrane recycling.