Membrane and cytoplasmic proteome biology of anterior mice hippocampus

The prevalence of neurodegenerative diseases increases steadily and proteomics on disease-related brain areas become more and more complex, whereas suitable samples are scarce. Due to this difficulty, animal models are indispensable in understanding human biology and disease, and the most commonly used model is the mouse (Mus musculus). Mice share many genetic and physiological characteristics with humans, breed rapidly, and can be genetically modified leading to the functional characterisation of many proteins. As for human, the mouse hippocampus is one of the most important areas of the brain and has therefore been an object of intensive investigation for many years. Mouse hippocampus has served as models for degenerating brain diseases associated with Alzheimer's disease, Parkinson's disease, ischemia, epilepsy, synaptic plasticity and underlying mechanisms of learning and memory.

Using 2 anterior parts of normal mouse hippocampi, we intend to characterize membrane and cytoplasmic proteome using a modified procedure previously established (WISNIEWSKI et al., 2008). This method permit the preparation and analysis of 2 individual fractions enriched in membrane and cytoplasmic proteins. The method for separation of membrane and cytoplasm fractions comprises a stepwise depletion of non-integral membrane proteins from entire tissue homogenate by high-salt, carbonate, and urea washes. The cytoplasmic proteins obtained from high-salt depletion are considered as the soluble fraction. The membrane proteins obtained from the stepwise depletion are considered as the insoluble fraction. Enzymatic digestion of both membrane and cytoplasmic fractions are carried out without use of detergents by double digestion with endoproteinase Lys-C and trypsin. Digested peptide fractions are loaded on StageTips for rapid desalting and are separated by online reversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC). Separated peptides are analyzed by electrospray tandem mass spectrometry (HPLC-ESI-MS/MS).

The entire procedure allows rapid processing and preparation of samples from minute amounts as 30-40 mg frozen tissue leading to about 70 g of membrane proteins and 500 g of cytoplasmic proteins. This can be extremely helpful for proteomic profiling of small pieces of tissue and clinical material.

Analyses of membrane fraction identified about 45% total membrane proteins. This fraction includes glutamate receptor 1 and 2, proteins involved into the trafficking of synaptic vesicles, lipid-anchor proteins, G proteins involved in various transmembrane signalling systems, NMDAR signalling complex proteins, voltage channel proteins… Analyses of the cytoplasmic fraction identify various proteins belonging to different compartments and/or pathways. A large amount of these proteins are specifically expressed into the hippocampus and/or into the nervous system.