Vibrational spectroscopy with metasurface optics (VISMO) for high-throughput identification of protein post-translational modifications

Project Summary/Abstract Proteins are critical to a wide array of cellular processes - from cell signaling to immune responses, nutrient transport, growth, and metabolic regulation. Proper function of proteins depends sensitively on the type, location, and abundance of post-translational modifications (PTMs). Aberrations in PTM properties can degrade protein function and compromise the physiology and function of the cells in which they reside. Indeed, altered PTMs have been implicated in a variety of diseases, including cancers, auto-immune diseases, neurodegenerative diseases, and inflammation. Nucleic acid sequencing gives almost no insight into PTM attributes, and sensitive and high-throughput characterization and quantification of PTMs remains a daunting challenge for mass spectroscopy (MS) and immunoassays. Pumpkinseed is developing an optics-based and label-free approach to measure protein PTMs, termed Vibrational Spectroscopy with Metasurface Optics (VISMO). VISMO offers high-speed analysis of millions of proteins – including their sequence, structure, and interactions – with three key advantages over other proteomics solutions: (1) In a label-free format, we uniquely identify each molecule via its vibrational scattering spectra, rather than relying on lower-resolution fluorescent tagging. (2) Using nanostructured Si chips, we create highly efficient optical resonators that both dramatically amplify the signal-to-noise ratio – even for very low abundance biomarkers – and increase sample throughput, with simultaneous measurement of ~3 million proteins per square centimeter. (3) We use cutting-edge machine learning (ML) algorithms to dissect information-rich features from the vibrational spectra, including the wavenumbers that correspond to the primary, secondary, and tertiary structure of the protein, as well as changes related to protein or small-molecule binding. Our Phase I proposal will develop this revolutionary platform for sensitive, high-throughput, and high-resolution protein detection, including their abundance, sequence, and PTMs. Aim 1 develops sensors that enable sensitive, few-to-single-molecule spectroscopy, builds our catalog of spectra for particular amino acids, short peptide sequences, and PTMs; and develops machine learning (ML) algorithms that provide interpretability to each molecule’s optical scattering signature. Aim 2 advances our methods for protein sequencing using chemical cleavage and our patented ‘sequencing by subtraction’ methodology. Phase I results will form the foundation of our Phase II proposal, with potentially far-reaching impacts on whole-proteome sequencing and protein-profiling of the vast repertoire of cell types. Biotherapeutic companies and academic researchers studying intact, native proteins will value the high-resolution that Pumpkinseed’s platform offers compared to existing ELISA-type assays, and the higher-sensitivity and throughput compared to traditional mass spectrometry. Our approach provides a new methodology for probing protein structure and interactions in order to reveal new dimensions of disease etiology and distinct opportunities for new therapeutic development.