Neuroproteomics

Synapses
Synapses form the nuts and bolts of the brain; they are essential cellular elements governing the majority of communication in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation of transmitter receptors and signal transduction cascades. It is generally believed, that neuronal activity-dependent change of synaptic efficacy (synaptic plasticity) is at the basis of learning and memory and is encoded by sequential molecular events at the synapse. In particular, synaptic activity elicits signal transduction that drives downstream events, notably alteration of protein-protein interaction and protein trafficking, changing protein constituents in distinct synaptic sub-domains and consequently alteration of synapse function. Aberrant changes in the synapse proteome have been implicated in various psychiatric disorders.

From single proteins to a systems biology approach
Studies of the nervous system are often focused on single to a few genes or proteins. However, this does not yield sufficient insight into the mechanisms by which neural processes, for instances in synapses, are accomplished or how they are deregulated. Synaptic function depends on the coordination of extended series of molecular events, of complex protein interactions, and less on the properties of single molecules. In order to allow novel insight into systems (dys)function, we have developed and implemented a quantitative proteomics technology that has the analytical power to unravel important clues to the (patho-)physiological processes leading to the observed (behavioral) phenotypes.

A proteomics workflow
We have established a quantitative proteomics workflow. This consists of a stable isotope tag (iTRAQ reagents) labelling of samples of interest for relative quantitation of up to 8 samples in a single experiment. This allows differential quantitation across experiments and with high statistical power. The characterization and quantitation are carried out by tandem liquid chromatography-MS/MS (using a MALDI tof/tof MS, the 5800 Proteomics Analyser from AB). Using this approach we can detect global regulation of functionally related protein classes, which allows generation of hypothesis to explain the observed (altered) phenotype.

Interaction proteomics
We are pursuing interaction proteomics, using hundreds of antibodies raised across different classes of synapse proteins. For this we use affinity purified protein complexes, which are separated on SDS-PAGE, digested and analysed by nanoLC-MS/MS (using a ESI-MS, the LTQ-orbitrap from Thermo). This study is accompanied by two large-scale analyses employing blue-native gel electrophoresis and proximity ligation assay, respectively, to confirm and localize the protein-protein interaction. The study is aimed at identifying the protein network (interactome) of the synapse, and to translate this into realistic dynamic models of synapse function. It is part of the EU-FP7 SynSys consortium.