PROTEOMICS  Proteomics is the large scale study of proteins within an organism. For HD, this is a technique we use to define new interactions with the huntingtin protein that are relevant to understand how huntingtin works, how huntingtin is toxic when polyglutamine expanded, and define new targets for drug targeting for HD. The basis for this work is protein affinity chromatography, a technique in which we use fragments of huntingtin as ligand for purification of proteins from a cellular extract. This is a commonly used technique to define interactors with proteins, however we have adapted this technique to help define true interactors from the very high noise that is inherent to this method, by the use of: 1. varying ligand protein concentrations 2. fractionated extracts 3. control ligands with subtle mutations defined in vivo. 4. different elution conditions Re-iterative Protein Micro Affinity Chromatography and Validation of Direct Interacting Proteins. A. Pure Htt Exon1 Q16 or Q46 is covalently linked to active ester agarose matrix and packed into a 20ul microcolumn for chromatography with 500ml of mouse whole cell brain extracts or membrane detergent extracts under physiological salt concentration. After loading and washing, proteins and complexes are eluted under high salt conditions to remove proteins from the affinity column and disrupt higher order complexes. Fractions are dialysed back to physiological salt concentrations and re- chromatographed over a new column. After a second load and wash, Final elution is salt step gradient, followed by high salt and 0.1% SDS across columns of varying ligand concentrations. Liquid eluates are then analysed by mass spectrometry to identify proteins (LC-MS-MS). B. Identified proteins will be expressed and purified from E.coli and passed over ligand columns, or placed into reciprocal co-immunoprecipitation experiments to determine direct interactions with no other factors present, at this point, affinity constants can be measured. C. Protein cDNAs will be fused to AFP variants and expressed in culture cells, either primary neuronal or other, for fluorescent microscopy-based assays. D. Interactions will be further defined using site-directed mutagenesis of the polyglutamine disease protein, effects of cell biological assay drugs like leptomycin B and cell biology stains for ER, DNA, RNA, lysosome, endosome, mitochondria, as well as long time course measurements of dynamics and short measurements by FRAP and FRET. (click on diagram to enlarge)