Massively parallel single-molecule protein quantitation using protein identification by short-epitope mapping (PrISM)

Introduction: Analyzing protein samples at single-molecule resolution holds the promise of achieving improved quantitative dynamic range, reproducibility, and sensitivity. By employing multi-affinity probes to interrogate immobilized, intact proteins, we aim to use Protein Identification by Short-epitope Mapping (PrISM) to analyze >95% of the proteome with broad dynamic range. PrISM has the potential to revolutionize proteomics by delivering highly accurate, large-scale analyses of protein samples.

Methods: Proteins were conjugated to DNA nanoparticles and deposited on a high-density patterned flow cell at optically resolvable locations. PrISM utilizes non-traditional affinity reagents called multi-affinity probes, designed with high affinity and low specificity in order to bind short epitopes present in multiple proteins across the proteome. Multi-affinity probes were applied to proteins to generate binding patterns for each single-molecule protein, which are translated to protein identifications and quantities using machine learning. We acquired PrISM data on a human osteosarcoma cell line (U2OS), protein mixtures, and control samples using hundreds of multi-affinity probes.

Results: We demonstrate detection and quantification of proteins from the U2OS cell line using PrISM data comprising billions of single-molecule binding measurements per sample. The detected proteins span a wide dynamic range of abundance in the U2OS cell line sample. We compare PrISM protein identification to mass spectrometry analysis of the same cell line. Improvements in instrumentation, reagents, and data processing approaches yield improved quantitative performance defined by sensitivity, accuracy, reproducibility, and dynamic range. An in-depth analysis of binding data shows a high diversity among patterns generated and indicates an opportunity to identify more proteins by adding more multi-affinity probes.

Conclusions: Steady improvement in the Nautilus platform driving the PrISM approach has resulted in detection of proteins from a human cell line sample at single-molecule resolution.