Proteins are responsible for much of the structure and function of all cells. Subtle modifications of various pro- teins, i.e., post-translational modifications (PTMs), can be made by cells to profoundly affect the function or structure of the protein. For example, common modifications such as phosphorylation can turn some proteins âoffâ or âonâ, acetylation can alter chromatin-binding proteins to change DNA structure and activity, and methyl- ation plays a role in activating numerous enzymes involved in gene regulation. Particular amino acid modifications have been identified at precise residues on only select proteins, in which cases antibodies can be generated to recognize them with high sensitivity and specificity. Although analysis by mass spectrometry can identify the presence and often the site of such modifications, a large quantity of sample is needed, and it is difficult or impossible to define whether multiple modifications are present on a single protein molecule. These approaches do not allow for single-molecule detection of PTMs on multiple residues. Glyphic Biotechnologies was formed to commercialize a novel strategy to sequence individual protein molecules in their entirety. This process is based on ligating the first (N-terminal) amino acid to a linker molecule called CP, which enables isolation and highly sensitive identification of the N-terminal amino acid. The process is repeated for each subsequent amino acid, yielding the protein sequence. The approach has the potential to simultaneously sequence millions to billions of individual protein molecules in hours. Developing this technology will revolutionize protein analysis by making large-scale protein sequencing feasible, inexpensive, and routine. The current proposal focuses on developing reagents specifically to detect amino acids containing three specific PTMs, allowing them to be sequenced with this technology. In Aim 1, we will generate antibodies to recognize PTMs linked to CP, allowing us to detect those modified amino acids in the sequencing reaction. In Aim 2, we will further optimize the antibodies and demonstrate the feasibility of using them to sequence individual proteins with PTMs among a background of non-modified proteins. Success of these Aims will enable the prospective Glyphic protein sequencing platform to detect and quantify PTMs in complex protein mixtures without any prior knowledge of their identity or even their existence. When commercialized, it will enable clinical diagnosis of disease based on the level of known PTMs in a patient sample. Moreover, it will allow identification of unique PTMs to develop additional tests for as-yet unknown biomarkers.
Public Health Relevance Statement: NARRATIVE No current technology is capable of unbiased identification of post-translational modifications (PTMs) in complex protein samples. Glyphic Biotechnologies is developing a novel method of single-molecule protein sequencing, analogous to âNext-Generationâ Sequencing of DNA. In conjunction with this technology, Glyphic proposes here to develop reagents to specifically detect PTMs for applications in clinical diagnostics and basic research.
Project Terms: Acetylation; Affect; Primary Protein Structure; protein sequence; Amino Acid Sequence; aminoacid; Amino Acids; Antibodies; Clinical Treatment Moab; mAbs; monoclonal Abs; Monoclonal Antibodies; Biological Assay; Assay; Bioassay; Biologic Assays; Biotechnology; Biotech; Cells; Cell Body; Chemistry; Chromatin; Disease; Disorder; Dyes; Coloring Agents; Engineering; Enzyme-Linked Immunosorbent Assay; ELISA; enzyme linked immunoassay; Enzymes; Enzyme Gene; Future; Gene Expression Regulation; Gene Action Regulation; Gene Regulation; Gene Regulation Process; Human; Modern Man; Libraries; Ligation; Closure by Ligation; Lysine; L-Lysine; Methods; Methylation; Fluorescence Microscopy; Fluorescence Light Microscopy; Mus; Mice; Mice Mammals; Murine; Noise; Parents; parent; Patients; Peptides; Phosphorylation; Protein Phosphorylation; Post-Translational Protein Processing; Post-Translational Modification Protein/Amino Acid Biochemistry; Post-Translational Modifications; Post-Translational Protein Modification; Posttranslational Modifications; Posttranslational Protein Processing; Protein Modification; Proteins; Oryctolagus cuniculus; Domestic Rabbit; Rabbits; Rabbits Mammals; Reagent; Research; Sensitivity and Specificity; Signal Transduction; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Specificity; Mass Spectrum Analysis; Mass Photometry/Spectrum Analysis; Mass Spectrometry; Mass Spectroscopy; Mass Spectrum; Mass Spectrum Analyses; Technology; Testing; Yeasts; Phosphotyrosine; Tyrosine-O-phosphate; Measures; Diagnostic tests; Titrations; Peptide Domain; Protein Domains; Tertiary Protein Structure; Blood Sample; Blood specimen; Label; improved; Site; Area; Surface; Solid; Phase; Link; Chemicals; Individual; Ligand Binding Protein; Ligand Binding Protein Gene; Protein Binding; bound protein; Binding Proteins; polyclonal antibody; clinical diagnosis; Diagnostic; Knowledge; Hour; Protocols documentation; Protocol; Reaction; Techniques; success; Fluorescence Resonance Energy Transfer; FRET; Förster Resonance Energy Transfer; Surface Plasmon Resonance; Structure; immunological diversity; novel; member; Basic Science; Basic Research; Eukaryota; Eukaryote; Peptide Sequence Determination; Amino Acid Sequence Determinations; Protein Sequence Determinations; Protein Sequencing; Protein Sequencing Molecular Biology; Proteome; Sampling; cross reactivity; Proteomics; single molecule; Molecular Interaction; Binding; protein complex; protein structure; protein structures; proteins structure; Affinity; DNA Structure; Detection; Diagnostics Research; Protein Analysis; Resolution; resolutions; Process; Modification; Image; imaging; computational studies; computer studies; next generation; new approaches; novel approaches; novel strategy; novel strategies; pathogen; prospective; NH2-terminal; N-terminal; innovate; innovative; innovation; clinical relevance; clinically relevant; commercialization; tumor; bio-markers; biologic marker; biomarker; Biological Markers; disease diagnosis; NGS Method; NGS system; next gen sequencing; nextgen sequencing; next generation sequencing; sequencing platform; identification of biomarkers; marker identification; biomarker identification; circulating markers; circulating biomarkers; medical diagnostic; clinical diagnostics; DNA seq; DNAseq; DNA sequencing; Visualization; diagnostic
