Single molecule peptide sequencing methods

A method of polypeptide sequencing comprising:

• (a) contacting a polypeptide with a non-optimized plurality of low-affinity amino acid binders to allow binding of one or more binders of the non-optimized plurality to a N-terminal amino acid of the polypeptide, wherein the binding produces a specific detectable signal;
• (b) identifying, based on the specific detectable signal, the N-terminal amino acid of the polypeptide, thereby generating an identified N-terminal amino acid;
• wherein identifying the N-terminal amino acid of the polypeptide comprises:
• (i) kinetically measuring the specific detectable signal produced by the binding of the one or more binders to the N-terminal amino acid of the polypeptide, thereby generating kinetic measurements for the one or more binders;
• (ii) combining the kinetic measurements;
• (iii) determining a binding profile of the non-optimized plurality of low-affinity amino acid binders based at least in part on the combined kinetic measurements;
• and
• (iv) identifying the N-terminal amino acid of the polypeptide from the binding profile of the non-optimized plurality of low-affinity amino acid binders;
• (c) removing the identified N-terminal amino acid of the polypeptide thereby exposing a subsequent N-terminal amino acid of the polypeptide;
• and
• (d) repeating steps (a) through (c) to identify the subsequent N-terminal amino acid of the polypeptide.