Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures

The identification and quantification of proteins lags behind DNA sequencing methods in scale, sensitivity and dynamic range. Here we show that sparse amino acid sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescent labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor reductions in each molecule’s fluorescence following consecutive rounds of Edman degradation. The obtained sparse fluorescent sequence of each molecule was then assigned to its parent protein in a reference database. We demonstrate the method on synthetic and naturally-derived peptide molecules in zeptomole-scale quantities. We also fluorescently label phosphoserines and demonstrate single-molecule, positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should empower studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single molecule sequencing.