Single-molecule sequencing of post-translational protein modifications

SUMMARY 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 “off” or “on”, 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.