ABSTRACT Despite the wealth of accurate and high sensitive data from next-gen DNA sequencing technologies, understanding the impact of the observed genomic mutations or alterations in the transcript (such as from splicing or transcriptional errors) on the protein structure and function is often unclear. To complicate this understanding are observations, where small abundant proteoforms can cause toxic intracellular aggregates. This link between genome/transcriptome to protein structure is unclear, because methods for characterizing protein structures often involve recombinant protein production and the use of analytical techniques that perform ensemble analysis. In this proposal, we introduce Fluorescent Protein Painting, a workflow for obtaining protein’s structural profiles by expanding the Erisyon’s existing fluorosequencing (single molecule protein sequencing) technology. The steps in workflow are - (a) reversible, covalent capture of natively folded proteins, (b) fluorescently labeling select surface exposed residues, (c) releasing, denaturing and digesting the proteins before (d) reacting the internal residues amino acid residues with second set of fluorophores. Performing fluorosequencing on this population of peptides will result in generation of protein structural profile or foot-prints on millions of individual peptides. Since the proteins are directly obtained from their native environment and the method can discriminate different protein conformations at single molecule sensitivity, we will have a better understanding of the impact of genomic mutations on the functional state of the biological sample.