Neuroproteomics is the study of the proteome, or the collection of proteins encoded by the genes of an organism, in particular that of the central nervous system (CNS). With the development of new techniques and the improvement of those already available, it is now possible not only to identify proteins, but also to determine changes in their abundance under various conditions (1
). This is particularly useful in understanding the physiological function of biological systems, as well as determining the functional implication of alterations in proteins in disturbed states, such as those induced by neurodegenerative disorders and/or drugs of abuse (2
In the CNS, synapses are essential for the communication between neurons. Upon stimulation, a presynaptic neuron releases neurotransmitters that bind to receptors in the postsynaptic neuron, which in turn induces a series of events in response to the stimulus. One of the most fascinating properties of the CNS is synaptic plasticity, or the ability to reconfigure and/or modulate synapses to accommodate for the wide variety of stimuli that they receive at any given time. The inability to respond adequately may lead to the development of neurodegenerative or addictive disorders.
Neuroproteomic studies have started to identify the proteins present in different compartments of the synapse, including synaptosomes (3
) and presynaptic and postsynaptic terminals (4
), mainly through subcellular fractionation protocols. This type of approach facilitates the analysis by reducing the complexity of the system. Moreover, it enriches synaptic compartments with less abundant proteins, which are commonly masked by those with the highest abundance. One way to take advantage of this methodology is to select a brain region, isolate the synaptic fraction of interest before and after a treatment (such as exposure to morphine), and identify the proteins in the fraction as well as their relative changes upon treatment (5
The approach described in this chapter is divided into two major sections. Section 2 describes the details for isolating a brain region and separating it into synaptic fractions, using subcellular fractionation. It is expected that (at least) two samples that undergo separate treatments (such as exposure to morphine and a control) are prepared. Section 3 details the steps required to isolate proteins from the fraction, digest them, differentially label them with appropriate isotopic labels, and perform mass spectrometry to identify the peptides and hence the proteins from the original sample. Details on data analysis are also covered, which results in the generation of a list of proteins and the relative change in protein levels upon treatment. While we have used specific examples from our research such as working with synaptic proteins after animal exposure to morphine, these protocols are general enough to work with other animal models, other brain regions, and a range of treatments. In addition, while specific fractionation and mass spectrometric equipment have been used, other instrumental platforms can be used with appropriate modifications.