Single-Molecule Protein Identification Through Peptide Chain Barcoding and Optical Readout

Abstract

A method for single-molecule protein identification and post-translational modification (PTM) mapping could potentially allow high-sensitivity detection and profiling of the cellular and circulatory proteome, and helps to better characterise cell types and differentiation states, analyse cellular regulatory programs and dynamics changes. Current workhorse approach of mass spectrometry based proteomic methods rely on digestion of proteins into short peptide fragments, exhibit biased fragment ionisation and have limited detection sensitivity. Combined with recent development in super-resolution single-molecule fluorescence microscopy methods, we are developing a novel method for single-molecule protein identification through peptide chain barcoding and optical readout. In particular, we will apply previously developed DNA-PAINT (point accumulation for imaging in nanoscale topography) and DMI (discrete molecular imaging) fluorescence super-resolution methods to protein molecules that are residue-specifically labelled with oligonucleotide probes. Using a combination of high-resolution (<5 nm) single-molecule imaging and blinking kinetics analysis, we present bioinformatics analysis demonstrating discrimination and identification power of our method, as well as high-efficiency residue labelling and single-molecule optical identification of short model proteins.