Label‐Free YaxAB Nanopore Fingerprinting of Cancer‐Associated Single‐Residue Bcl‐xL Variants

Resolving cancer-associated protein variants remains difficult when variants share similar molecular masses and folded structures. Conventional assays often require labels or provide ensemble-averaged readouts, limiting their ability to resolve subtle variant differences. Here, a YaxAB nanopore workflow is presented for label-free single-molecule current fingerprinting of wild-type Bcl-xL and the cancer-associated single-residue variants N136K and R165W. Individual Bcl-xL proteins were electro-osmotically trapped within the funnel-shaped YaxAB lumen, where they generated characteristic ionic-current events. These events formed reproducible, analyte-specific event populations in normalized current blockade and current noise space, resolving wild-type Bcl-xL, N136K, and R165W as distinct single-molecule current fingerprints. Wild-type Bcl-xL and N136K remained distinct despite a mass difference of only 14 Da, and the fingerprint separation was consistent with combined differences in excluded volume, net charge, and dipole moment. Concentration-dependent capture-rate analysis further supported sub-nanomolar to low-nanomolar detection corresponding to protein amounts in the sub-picomole to low-picomole range, while capture-rate normalization brought the estimated concentrations closer to the nominal concentrations. These results present YaxAB nanopore sensing as a label-free single-molecule workflow for the current fingerprinting of cancer-associated single-residue Bcl-xL variants, with low-concentration detection as a supporting analytical capability.