Project Grant R01GM165889

The National Institute of General Medical Sciences awarded the University of Washington $2,226,244 on September 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to develop nanopore technology for single-molecule protein sequencing of intact proteoforms.

The funded work advances unfoldase-coupled nanopore arrays to directly sequence native proteins at single-molecule resolution, detect post-translational modifications, and analyze complex proteoforms. The project addresses current limitations in throughput, sample compatibility, dataset scale, and performance on large proteins beyond existing proteomics methods such as mass spectrometry and antibody-based approaches. The technology uses the ClpX unfoldase enzyme to translocate intact proteins through engineered nanopore sensors, generating ionic current signatures that identify amino acids and modifications without requiring fragmentation or inference steps. Planned deliverables include optimized free-running unfoldase nanopore sequencing through ClpX stalling control and analyte barcoding; scaled sequencing datasets using barcoded peptide and proteoform libraries to train machine learning models for amino acid and post-translational modification calling; and validation and benchmarking on BRCA1, a clinically significant multi-domain protein with extensive proteoform diversity.

The University of Washington Office of Sponsored Programs, acting as prime recipient, will perform the work in Seattle, Washington, with period of performance from September 1, 2026, through August 31, 2030.