Single-Molecule Proteomics using Protein Identification by Short-epitope Mapping

The field of proteomics is poised for a single-molecule revolution – enabling more comprehensive analysis of the proteins in a sample with increased sensitivity, reproducibility, and accessibility. Here we demonstrate Protein Identification by Short-epitope Mapping (PrISM). PrISM is a single-molecule analysis method where intact proteins are immobilized, iteratively probed with multi-affinity probes, and machine learning is applied to convert binding patterns to protein identification and quantification. PrISM uses non-traditional affinity reagents with high affinity and low specificity that bind to short epitopes in multiple proteins. Simulations using these multi-affinity probes show that the accumulated information from multiple rounds of detection of short (2-4 amino acid) epitopes enables identification of more than 95% of the human proteome with just 300 probes. Model proteins were conjugated to DNA nanoparticles and deposited on a high-density patterned flow cell at optically resolvable locations. We acquired PrISM data using >30 multi-affinity probes targeting trimer or tetramer sequences. We demonstrate identification of individual protein molecules through iterative probing with our multi-affinity probes. By combining single-molecule analysis, intact (non-digested) proteins, and iterative probing, PrISM provides a new tool for the quantitation of proteins enabling new exploration of the proteome.

 

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