Technology for Identification of Epiproteomes in Tissue Samples

Univ Of Arkansas For Med Scis, Little Rock AR

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Abstract

? DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a cutting-edge technology for identifying epiproteomes in tissue samples, which will provide an approach that will redefine how NIDA researchers study epigenetic responses to drug abuse/addiction. We define an epiproteome as the proteins, histones and histone posttranslational modifications at a defined ~1 kb chromosomal location. In previous work, we have developed a groundbreaking technology platform called ChAP-MS (Chromatin Affinity Purification with Mass Spectrometry) that allows us to biochemically isolate a ~1 kb section of a chromosome and identify the epiproteome using high resolution proteomics. Our ChAP-MS platform provided for the first ever isolation of a specific, native chromatin locus for proteomic analysis. The first and second generation ChAP-MS technologies used ectopic expression of affinity probes for chromatin isolation and were designed for cell culture studies; thus, precluding the analysis of animal models. As outlined in this proposal, we will develop a third generation approach called recombinant rCRISPR-ChAP-MS in yeast and translate it to brain tissue samples. The third generation approach will circumvent ectopic expression of affinity probes; thus, enabling the analysis of tissue. We envision the long term application of the rCRISPR-ChAP-MS approach in defining epigenetic mechanisms regulating drug abuse/addiction. Specific Aim 1 will focus on developing rCRISPR-ChAP-MS for application to tissue samples. Using S. cerevisiae as a model system in Aim 1, we will develop rCRISPR-ChAP-MS by analysis of epiproteomes at the GAL1 promoter under transcriptionally active and repressed conditions. Specific Aim 2 will focus on translating rCRISPR-ChAP-MS to brain tissue. In Aim 2, we will target our optimized approach to the gene promoter of AMPA receptor subunit GluA1 in striatum from rats chronically exposed to methamphetamine.