Sunday, May 11, 2025
5/11/2025
HistoneScan?: a multiplex immunoassay for histone epigenetic profiling - ABSTRACT Gene activity is controlled by histone proteins that interlink with DNA molecules to form condensed chromatin structures. Changes in these histones are thus associated with dysregulated biological processes and disease. To date, DNA technologies lack biochemical tests proficient in detecting two or more histone modifications within the same molecule. The use of histone-specific antibodies confers detection of a limited number of histone modifications and thus complex modification patterns are not picked up effectively. Cross-reactivity occurring within similar protein sequences is an additional limitation of these current assays. In aims of enhancing detection, identification and quantification of complex human histone modifications, we propose the development of a multiplex analytical assay HistoneScan™. HistoneScan™ technology involves initial generation of histone fragments that comprise the histone modification of interest by means of sample digestion. Antibodies immobilized to specific beads are then used to capture modified proteins via a technique known as single bead immunoaffinity capture; and incorporation of several beads for one antibody allows for assay reproducibility. Beads are then aligned onto a BAMS™ slide to enable peptide extraction from their respective beads for analysis by mass spectrometry; by which data analysis determines relative proportions of specific histone modification combinations. In this research project, we propose to develop antibody-bead probes for capture of up to 100 modifications within human histone proteins H1, H2A, H2B, H3 and H4 and to develop a histone extraction and digestion technique for optimized HistoneScan™ workflow. Moreover, we aim to enhance data reporting by development of an analytical software, HistoneView™, and the validation of HistoneView™ as a screening technique to be used for chromatin profiling applications. This technique overrides the main disadvantage of current assays, i.e., Western blots; that are designed to only measure single protein changes and shifts to revealing co-occurring histone modifications in a high-throughput and cost-effective direct approach. The additional use of BAMS™ technology will also allow for the detection of histone modifications that typically serve as useful disease biomarkers, such as histone-modifying enzymes and thus pioneer the way forward in personalized medicine.
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