Single molecule peptide sequencing methods
The invention, in part, includes methods of single molecule protein sequencing that include using weak binding spectra in the amino acid identification.
1. 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.
2. The method of claim 1 , further comprising repeating steps (c) and (d) a sufficient number of times to identify a portion or all of an amino acid sequence of the peptide.
3. The method of claim 1 , wherein the specific detectable signal is a fluorescent signal, a luminescent signal, or an electrical signal.
4. The method of claim 1 , wherein the detectable signal is detected using a single-photon avalanche diode (SPAD) detection method.
5. The method of claim 1 , further comprising determining specific detectable signals produced by binding of the one or more binders to the N-terminal amino acid and the subsequent N-terminal amino acid after removing the identified N-terminal amino acid of the polypeptide in (c).
6. The method of claim 5 , wherein determining the specific detectable signals comprises an optical detection method.
7. The method of claim 6 , wherein the optical detection method comprises microscopy.
8. The method of claim 5 , wherein determining the specific detectable signals comprises an electrical detection method.
9. The method of claim 8 , wherein the electrical detection method comprises a single-photon avalanche diode (SPAD) detection method.
10. The method of claim 5 , wherein determining the specific detectable signals produced by the binding of the one or more binders to the N-terminal amino acid occurs simultaneously.
11. The method of claim 5 , wherein determining the specific detectable signals produced by the binding of the one or more binders to the subsequent N-terminal amino acid occurs simultaneously.
12. The method of claim 1 , wherein the kinetically measuring comprises detecting a plurality of time-averaged specific detectable signals of the non-optimized plurality of low-affinity amino acid binders that bind the N-terminal amino acid of the polypeptide.
13. The method of claim 12 , wherein the detecting the plurality of time-averaged specific detectable signals comprises determining a length of time of the binding events of the non-optimized plurality of low-affinity amino acid binders that bind the N-terminal amino acid of the polypeptide.
14. The method of claim 1 , wherein the kinetically measuring comprises measuring using a high time resolution capable of detecting individual binding and unbinding events.
15. The method of claim 1 , wherein the kinetically measuring comprises measuring using a low time resolution capable of detecting and integrating signals of many binding and unbinding events to generate a plurality of detected time-averaged signals of the many binding and unbinding events, wherein a binding affinity is deduced based on the detected time-averaged signals of the many binding and unbinding events.
16. The method of claim 1 , wherein removing the identified N-terminal amino acid from the polypeptide comprises a cycle of Edman degradation.
17. The method of claim 1 , further comprising simultaneously detecting a plurality of produced specific detectable signals.
18. The method of claim 1 , wherein the non-optimized plurality of low-affinity amino acid binders comprises at least 5, 10, 15, 20 or more different binders.
19. The method of claim 1 , wherein each of the non-optimized plurality of low-affinity amino acid binders is a low-specificity binding reagent.