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Comprehensive understanding of a gene's expression and regulation at the molecular level requires identification of all proteins interacting with the gene. HyCCAPP (Hybridization Capture of Chromatin Associated Proteins for Proteomics) is an approach that uses single-stranded DNA oligonucleotides to capture specific genomic sequences in cross-linked chromatin fragments and identify associated proteins by mass spectrometry. Previous studies have shown HyCCAPP to provide useful information on protein-DNA interactions, revealing the proteins associated with the GAL1-10 region in yeast. We present here a multiplexed version of HyCCAPP. Utilizing a toehold-mediated capture/release strategy, HyCCAPP is targeted to multiple genomic loci in parallel, and the protein binders at each locus are eluted in a programmable and selective fashion. Multiplexed HyCCAPP was applied to four genes (25S rDNA, ARX1, CTT1, and RPL30) in S. cerevisiae under normal and stressed conditions. Capture and release efficiencies and specificities were comparable to those obtained without multiplexing. Using mass spectrometry-based bottom-up proteomics, hundreds of proteins were discovered at each locus in each condition. Statistical analysis revealed 34-88 enriched proteins in each gene capture. Many of these proteins had expected functions, including DNA-related and ribosome biogenesis-associated activities. Multiplexed HyCCAPP provides a useful strategy for the identification of proteins interacting with specific chromatin regions.

Multiplexed Sequence-Specific Capture of Chromatin and Mass Spectrometric Discovery of Associated Proteins