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.
Public Health Relevance Statement: NARRATIVE This project aims to develop an analytical assay kit, which will detect, identify and quantify multiple co-occurring post-translational modifications to the proteins that support DNA and regulate gene transcription. These modifications are increasingly being used as disease biomarkers. Our technology will lead to a more robust method of identifying and validating these biomarkers for precision medicine.
Project Terms: Amino Acid Sequence; Primary Protein Structure; protein sequence; Antibodies; Biological Assay; Assay; Bioassay; Biologic Assays; Western Blotting; Immunoblotting; Western Immunoblotting; protein blotting; Cell Culture Techniques; cell culture; cell cultures; Cells; Cell Body; Chromatin; Data Analyses; Data Analysis; data interpretation; Data Reporting; data representation; Digestion; Disadvantaged; Disease; Disorder; DNA; Deoxyribonucleic Acid; Drug Controls; Enzyme-Linked Immunosorbent Assay; ELISA; enzyme linked immunoassay; Enzymes; Enzyme Gene; Genes; Regulator Genes; Transcriptional Regulatory Elements; regulatory gene; trans acting element; Hela Cells; HeLa; Histone H1; Histone H3; Histones; Human; Modern Man; Immobilization; orthopedic freezing; Immunoassay; Incidence; Lysine; L-Lysine; Methods; Esteroproteases; Peptidases; Protease Gene; Proteases; Proteinases; Proteolytic Enzymes; Peptide Hydrolases; Peptides; Post-Translational Modification Protein/Amino Acid Biochemistry; Post-Translational Modifications; Post-Translational Protein Modification; Posttranslational Modifications; Posttranslational Protein Processing; Protein Modification; Post-Translational Protein Processing; Proteins; Reagent; Research; Research Resources; Resources; Savings; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Signal Transduction; Software; Computer software; Mass Photometry/Spectrum Analysis; Mass Spectrometry; Mass Spectroscopy; Mass Spectrum; Mass Spectrum Analyses; Mass Spectrum Analysis; Technology; Testing; Time; Genetic Transcription; Gene Transcription; RNA Expression; Transcription; Generations; Measures; Data Set; Dataset; Custom; base; Site; Phase; Biological; biologic; Biochemical; screening tools; Screening procedure; Recovery; R-Series Research Projects; R01 Mechanism; R01 Program; Research Grants; Research Projects; Research Project Grants; Biological Function; Biological Process; Companions; Hour; Complex; Protocol; Protocols documentation; Slide; Pattern; Techniques; interest; Services; synthetic peptide; antibody conjugate; SAHA; Suberoylanilide Hydroxamic Acid; suberanilohydroxamic acid; Vorinostat; Reporting; Sampling; cross reactivity; Proteomics; histone modification; Address; Data; Detection; HDAC Agent; HDAC inhibitor; Histone deacetylase inhibition; Histone Deacetylase Inhibitor; Reproducibility; Chromatin Structure; Epigenetic Process; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Validation; Preparation; Process; Modification; Development; developmental; design; designing; cost effective; combinatorial; Biological Markers; bio-markers; biologic marker; biomarker; product development; ChIP-seq; ChIP Sequencing; chromatin immunoprecipitation-sequencing; screening; precision medicine; precision-based medicine; personalized medicine; personalization of treatment; personalized therapy; personalized treatment; epigenetic profiling; experimental study; experiment; experimental research
