Single molecule peptide sequencing methods

1. A method of identifying an N-terminal amino acid of a polypeptide, comprising:

(a) contacting a composition comprising a polypeptide with a set of independently selected N-terminal amino acid binding (NAAB) reagents, wherein a plurality of the independently selected NAAB reagents in the set bind to the polypeptide's N-terminal amino acid and the binding of each of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid produces a specific detectable signal;

(b) determining the specific detectable signal produced by the binding of each of the plurality of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid;

(c) kinetically measuring the determined specific detectable signals produced by the binding of the plurality of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid;

(d) combining the kinetic measurements;

(e) determining a binding profile of the set of independently selected NAAB reagents based at least in part on the combined kinetic measurements; and

(f) identifying the N-terminal amino acid in the polypeptide from the determined binding profile of the set of independently selected NAAB reagents.

2. The method of claim 1 , wherein the detectable signal is a luminescent signal, and optionally is a fluorescent signal.

3. The method of claim 1 , further comprising removing the N-terminal amino acid from the polypeptide to reveal a next N-terminal amino acid on the polypeptide, and repeating the steps (a)-(f) to identify the next N-terminal amino acid of the polypeptide.

4. The method of claim 3 , further comprising repeating the removal of the N-terminal amino acid and steps (a)-(f) a sufficient number of times to identify a portion or all of the polypeptide's amino acid sequence.

5. The method of claim 1 , wherein each of the independently selected NAAB reagents is a low affinity binding reagent for each of the polypeptide's amino acids.

6. The method of claim 1 , wherein each of the independently selected NAAB reagents is a low specificity binding reagent for each of the polypeptide's amino acids.

7. The method of claim 1 , wherein the kinetic measuring comprises detecting a plurality of time-averaged specific detectable signals of each of the independently selected NAAB reagents that bind the polypeptide's N-terminal amino acid.

8. The method of claim 7 , wherein detecting the time-averaged signal comprises determining a length of time of the binding events of the independently selected NAAB reagents that bind the polypeptide's N-terminal amino acid.

9. The method claim 1 , wherein the plurality of the independently selected NAAB reagents in the set comprises at least 5, 10, 15, 20 or more different binding reagents.

10. The method of claim 1 , wherein a means of determining the specific detectable signals produced by the binding of each of the plurality of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid comprises an optical detection method.

11. The method of claim 10 , wherein the optical detection method comprises microscopy, and optionally the microscopy comprises total internal reflection fluorescence (TIRF) microscopy.

12. The method of claim 11 , wherein the TIRF microscopy comprises kinetically measuring the determined specific detectable signals produced by the binding of the plurality of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid.

13. The method of claim 1 , wherein more than one detectable signal produced by the binding of each of the plurality of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid are simultaneously detected.

14. The method of claim 1 , wherein a high-dimensional vector of kinetically-measured affinities for the N-terminal amino acid is produced from the kinetic measurements.

15. The method of claim 1 , wherein the kinetically measuring comprises measuring using a high-time resolution measuring means capable of detecting individual binding and unbinding events.

16. The method of claim 1 , wherein the polypeptide is immobilized on a surface and optionally the polypeptide is immobilized in a manner to have, on average, no more than one peptide per a diffraction-limited spot.

17. The method of claim 3 , wherein a means for removing the N-terminal amino acid from the polypeptide comprises a cycle of Edman degradation.

18. The method of claim 1 , wherein each of the independently selected N-terminal amino acid binding reagents is derived from an independently selected aminopeptidase.

19. A method of determining an amino acid sequence of a polypeptide, comprising:

(a) contacting a composition comprising a polypeptide with a set of independently selected N-terminal amino acid binding (NAAB) reagents, wherein a plurality of the independently selected NAAB reagents in the set bind to the polypeptide's N-terminal amino acid and the binding of each of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid produces a specific detectable signal;

(b) determining the specific detectable signal produced by the binding of each of the plurality of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid;

(c) kinetically measuring the determined specific detectable signals produced by the binding of the plurality of the independently selected NAAB reagents to the polypeptide's N-terminal amino acid;

(d) combining the kinetic measurements;

(e) determining a binding profile of the set of independently selected NAAB reagents based at least in part on the combined kinetic measurements;

(f) identifying the N-terminal amino acid in the polypeptide from the determined binding profile of the set of independently selected NAAB reagents;

(g) removing the identified N-terminal amino acid to reveal a next N-terminal polypeptide; and

(h) repeating steps (a)-(g) on the next N-terminal amino acid of the polypeptide to determine a partial or full amino acid sequence of the polypeptide.

20. A method of spectral sequencing a peptide comprising:

(a) measuring one or more of probe-target binding affinities and probe-target binding kinetics of a plurality of low-affinity, relatively non-specific N-terminal-specific amino-acid binders (NAABs) and amino acid targets;

(b) collectively determining a spectrum of affinity across the NAABs, for each of the N-terminal amino acid targets;

(c) determining the identity of the N-terminal amino acids based on the collectively determined spectrum of affinity across the NAABs; and

(d) sequencing a peptide based on the determined identities of the N-termination amino acids.