Proteomique CAPM

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Membrane proteins assay by SPR

Membrane proteins located at the interface between subcellular compartments perform critical functions and are prime targets for pharmocologic agents. Though generally sparse, membrane proteins tend to be more abundant in some areas. Due to their hydrophobic property, quantification and analysis of membrane proteins using conventional biochemical techniques is difficult. Since its creation in 2000, the CAPM/Biocapteurs is specialized in these proteins study and has demonstrated that SPR technology constitute a method of choice for investigation of membrane proteins. By taking as model, one of the proteins of the membrane of synaptic vesicles, VAMP2 protein, we showed first that the presence of an acidic lipid environment influences the conformation and function of this protein (De Haro, PNAS 2004).

In addition we have developed a new investigative methodology in which intact synaptic vesicles are immobilized in a single step on biochips (Ferracci, Anal Biochem 2004). This "vesicle chip” configuration provides a simple setup for differential and/or functionnal proteomic of synaptic vesicle membrane components and can be adapted to the study of other types of organelles.

Based on SPR analysis of the immuno-capture rates of synaptic vesicles, we were also able to propose a highly sensitive new method for assaying membrane components without requiring solubilization and that can be performed on small samples from cultured neurons or brain slices (Ferracci, Biochem J 2005 ; Slezac, Neuron 2012).

One of the applications implemented on the platform is the assay of endoproteolytic activity of botulinum neurotoxins. Clostridal neurotoxins are the most toxic biological substances known. They inhibit synaptic neurotransmission by cleaving SNARE proteins. Their increasing use as therapeutic agents as well as their potential use in biological weapons justify further in vitro assays. Using native substrates, we have developed SPR assays to measure BoNT/A and B endoproteolytic activity (Ferracci, Biochem J 2005 ; Marconi, Toxicol Appl Pharmacol 2008). For BoNT/B, this test was shown to be 200 times more sensitive and up to 25 times faster than the reference in vivo toxicity test in mice and can detect the toxin in food or clinical samples (Ferracci, Anal Biochem 2011).

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