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US 2020/0400677 A1Published application
Single molecule peptide sequencing methods
AssigneeMassachusetts Institute of Technology
InventorsEdward BOYDEN, Adam Henry MARBLESTONE, Samuel Gordon RODRIQUES
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Abstract
The invention, in part, includes methods of single molecule protein sequencing that include using weak binding spectra in the amino acid identification.
Claims31
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.
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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.
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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.
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Dependencies are inferred from the claim text.