Proteomic analysis of synaptosomes using isotope‐coded affinity tags and mass spectrometry

Abstract

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.

Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development

“…For example, the cell body and axon terminal of the same neuron are often located in two different functional regions. Although some laboratories have demonstrated MudPIT as an efficient platform for high-throughput protein identification of the brain proteome Phillips et al 2004;Cagney et al 2005;Schrimpf et al 2005;Witzmann et al 2005), a large-scale quantitative methodology is lacking for analysis of the brain proteome. For example, using the AQUA (absolute protein identification) strategy, the differential expres-sion of 32 proteins between brain regions has been reported (Peng et al 2004;Cheng et al 2006).…”

Neuroproteomics – the tasks lying ahead

“…Despite recent progress, such as the enrichment of total plasma membrane from rat cerebellar tissue by affinity partitioning [100], further refinements are required to isolate and analyze the various plasma membrane microdomains in sufficient purity and yield. A recent proteomic analysis of synaptosomes isolated by differential and density-gradient centrifugation from mouse brain, resulted in more than 1100 protein identifications [132]. However, only 50% overlap was observed between two independent experiments and known synaptosomal proteins, including the inhibitory neurotransmitter receptors and vesicular glutamate transporters were not detected at all.…”

An Integrated Quantitative Proteomics and Systems Biology Approach to Explore Synaptic Protein Profile Changes During Morphine Exposure

“…Although these studies identified only a small collection of proteins relative to the thousands of proteins now known to occupy the excitatory PSD (Bayes and Grant, 2009), they were among the first to identify additional PSD-associated proteins vital to postsynaptic functioning, including an array of cytoskeletal and scaffolding proteins, cell-adhesion molecules, and proteins modulating small G-protein signaling, adaptor proteins, glutamate receptors, and various signaling molecules (Husi et al, 2000;Walikonis et al, 2000;Satoh et al, 2002). Building upon these early results, more recent investigations have utilized proteomic techniques ranging from SDS-PAGE and 2-DE coupled with mass spectrometry (Jordan et al, 2004;Li et al, 2004;Peng et al, 2004;Collins et al, 2006;Dosemeci et al, 2006;Klemmer et al, 2009), to ICAT labeling or MudPIT (Li et al, 2004;Yoshimura et al, 2004;Phillips et al, 2005;Schrimpf et al, 2005;Moron et al, 2007), and even tandem affinity purification (TAP) tagged-PSD-95 knock-in mice coupled with 1-DE and LC-MS/MS analysis (Fernandez et al, 2009) to identify thousands of proteins that are associated with the structure and function of the excitatory PSD (Collins et al, 2006;Fernandez et al, 2009;summarized in Table 2). Although the importance of these studies in elucidating the molecular and chemical components of the excitatory PSD cannot be overstated, only recently have the powerful tools Figure 1.…”