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

Abstract

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 GAL110 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.

HyPR-MS for Multiplexed Discovery of MALAT1, NEAT1, and NORAD lncRNA Protein Interactomes

“…The effect of release order on capture specificity and efficiency was previously evaluated and shown to not substantially impact these measurements. 49…”

Elucidating Protein–DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry

“…The beads were then concentrated into a smaller volume and washed once for 5 min and once for 1 h. To release the hybridized complexes, a toehold-mediated strand displacement strategy was employed (see Supplementary Information for details). This strategy was developed and demonstrated for the selective release of individual target loci from cross-linked yeast chromatin, 27,28 and here we extended the strategy to cross-linked human chromatin. First, alpha satellite release solution containing equimolar concentrations of the eight alpha satellite release oligonucleotides was added to the beads, and the resultant bead slurry was gently mixed for 15 min at room temperature.…”

Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

“…These probe–lncRNA-RBP complexes are isolated with streptavidin-coated magnetic beads [ 77 ]. After the simultaneous capture of multiple targets, each target is released in a sequence-specific manner, referred to as the toehold-mediated release [ 94 , 95 ]. Each probe harbors an additional 8-nt sequence, which does not hybridize the target, called the toehold sequence.…”