High-Rate Fingerprinting of Protein Isoforms by Quasi-regulated Enzyme-free Transport Through CytK Nanopores.

Nanopores are compelling tools for ultrafast single-molecule protein analysis. Recent investigations into both enzyme-free and enzyme-regulated transport mechanisms suggest that the faster kinetics of enzyme-free transport enable higher throughput, albeit requiring speed control for accurate protein characterization. Here we investigate the signals obtained from chemically unfolded proteins when transported through wild-type cytotoxin K (CytK) in an enzyme-free manner. We designed, expressed, and measured synthetic block isoforms derived from maltose-binding protein that contain repeat sub-sequences as benchmark molecules. Surprisingly, reverse capture of proteins (i.e., from the β-barrel side) proceeds with ~2.5 orders of magnitude higher rate than forward capture, allowing high-rate protein fingerprinting at sub-nM concentrations. We utilize high-voltage reverse capture for rapid analysis of the block isoforms, and found that the ionic current signal shapes, arising from stochastic movements with 4-7 amino-acid-long steps, reproducibly follow the number and order of sequence repeats. We corroborate our results with ionic current signatures obtained from molecular dynamics simulations, exhibiting matching signature shapes. Lastly, we demonstrate an example life-science research use case through rapid nanopore quantification of leaky and overexpressed proteins that contain short electrophoretic tags directly from crude bacterial lysate in under 30 minutes, which allows for rapid and selective quantification of full-length protein expression levels.