Methods of single-polypeptide sequencing and reconstruction

Patent metadata

Field	Value
Publication number	US-2021148922-A1
Application number	US-202017082940-A
Country	US
Kind code	A1
Filing date	Oct 28, 2020
Priority date	Oct 28, 2019
Publication date	May 20, 2021
Grant date	—

Abstract

Provided herein are methods of single-polypeptide sequencing and reconstruction. Also provided herein are compositions, kits and devices useful for the same.

First claim

1 . A method comprising:
(i) providing an enriched sample comprising a population of polypeptides; (ii) splitting the enriched sample into two or more subsamples; (iii) contacting each of at least two of the subsamples with a different modifying agent, wherein the modifying agent comprises a cleaving agent, thereby generating polypeptide fragments having a combination of cleavage patterns; and (iv) sequencing, in parallel, the polypeptide fragments, thereby determining the amino acid sequences of the polypeptide fragments; optionally wherein the polypeptide fragments generated in (iii) are combined into a single sample prior to the sequencing in (iv).

2 . The method of claim 1 , further comprising:
(v) reconstructing the sequences of polypeptides in (i) by aligning the amino acid sequences of the of the polypeptide fragments determined in (iv).

3 . The method of claim 2 , further comprising:
(vi) identifying or confirming the absence of polypeptide variants from the sequences of polypeptides reconstructed in (v).

4 . The method of claim 3 , wherein a polypeptide variant in (vi) comprises an alternative splice site, an amino acid insertion, an amino acid deletion, an amino acid substitution, and/or an amino acid chemical modification, optionally wherein the amino acid chemical modification is a post-translational modification.

5 .- 6 . (canceled)

7 . The method of claim 1 , wherein (i) comprises:
(a) providing a cell population; (b) lysing the cell population to generate a lysis sample comprising polypeptides expressed in the cell population; and (c) isolating a subset of the polypeptides from the lysis sample, thereby generating an enriched sample comprising a subset of the polypeptides expressed in the cell population.

8 . (canceled)

9 . The method of claim 7 , wherein (c) comprises:
i. contacting the lysis sample with a plurality of enrichment molecules, wherein at least a subset of the enrichment molecules in the plurality of enrichment molecules binds to a subset of the polypeptides in the lysis sample, thereby generating a bound subset of polypeptides and an unbound subset of polypeptides; and ii. isolating the bound subset of polypeptides or the unbound subset of polypeptides.

10 . The method of claim 9 , wherein:
each of the enrichment molecules in the plurality of enrichment molecules is an antibody, an aptamer, or an enzyme; or the enrichment molecules in a subset of the plurality of enrichment molecules comprise an antibody, an aptamer, or an enzyme.

11 . The method of claim 9 , wherein:
each of the enrichment molecules in the plurality of enrichment molecules is bound to a substrate; or the enrichment molecules in a subset of the plurality of enrichment molecules are bound to a substrate; optionally wherein the contacting of the plurality of polypeptides with the plurality of enrichment molecules occurs when the lysis sample comprising the plurality of polypeptides contacts the substrate.

12 . (canceled)

13 . The method of claim 11 , wherein the substrate is selected from the group consisting of a surface, a bead, a particle, and a gel, optionally wherein:
the surface is a solid surface; the bead is a magnetic bead; or the particle is a magnetic particle.

14 .- 18 . (canceled)

19 . The method of claim 1 , wherein the sequencing in (iv) comprises:
(a) contacting a polypeptide fragment with one or more terminal amino acid recognition molecules; and (b) detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with successive amino acids exposed at a terminus of the polypeptide fragment while the polypeptide is being degraded, thereby sequencing the polypeptide fragment.

20 . The method of claim 1 , wherein the sequencing in (iv) comprises:
(a) contacting a polypeptide fragment with a composition comprising one or more terminal amino acid recognition molecules and a cleaving reagent; and (b) detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with a terminus of the polypeptide fragment in the presence of the cleaving reagent, wherein the series of signal pulses is indicative of a series of amino acids exposed at the terminus over time as a result of terminal amino acid cleavage by the cleaving reagent.

21 . The method of claim 1 , wherein the sequencing in (iv) comprises:
(a) identifying a first amino acid at a terminus of a polypeptide fragment; (b) removing the first amino acid to expose a second amino acid at the terminus of the polypeptide fragment; and (c) identifying the second amino acid at the terminus of the polypeptide fragment, wherein (a)-(c) are performed in a single reaction mixture.

22 . The method of claim 1 , wherein the sequencing in (iv) comprises:
(a) contacting a polypeptide fragment with one or more amino acid recognition molecules that bind to the polypeptide fragment; (b) detecting a series of signal pulses indicative of association of the one or more amino acid recognition molecules with the polypeptide fragment under polypeptide degradation conditions; and (c) identifying a first type of amino acid in the polypeptide fragment based on a first characteristic pattern in the series of signal pulses.

23 . The method of claim 1 , wherein the sequencing in (iv) comprises:
(a) obtaining data during a polypeptide degradation process; (b) analyzing the data to determine portions of the data corresponding to amino acids that are sequentially exposed at a terminus of the polypeptide during the degradation process; and (c) outputting an amino acid sequence representative of the polypeptide.

24 . The method of claim 1 , wherein the sequencing in (iv) comprises:
(a) contacting a polypeptide fragment with one or more labeled affinity reagents that selectively bind one or more types of terminal amino acids at a terminus of the polypeptide fragment; and (b) identifying a terminal amino acid at the terminus of the polypeptide fragment by detecting an interaction of the polypeptide fragment with the one or more labeled affinity reagents.

25 . The method of claim 1 , wherein the sequencing in (iv) comprises:
(a) contacting a polypeptide fragment with one or more labeled affinity reagents that selectively bind one or more types of terminal amino acids at a terminus of the polypeptide fragment; (b) identifying a terminal amino acid at the terminus of the polypeptide by detecting an interaction of the polypeptide fragment with the one or more labeled affinity reagents; (c) removing the terminal amino acid; and (d) repeating (a)-(c) one or more times at the terminus of the polypeptide fragment to determine an amino acid sequence of the polypeptide fragment;
optionally wherein the method further comprises:
after (a) and before (b), removing any of the one or more labeled affinity reagents that do not selectively bind the terminal amino acid; and/or
after (b) and before (c), removing any of the one or more labeled affinity reagents that selectively bind the terminal amino acid.

26 .- 30 . (canceled)

31 . A method comprising:
(i) providing an enriched sample comprising a population of polypeptides; (ii) splitting the enriched sample into two or more subsamples; (iii) contacting each of at least two of the subsamples with a different modifying agent, wherein each modifying agent comprises a cleaving agent, thereby generating polypep

Assignees

• Quantum Si Inc

Inventors

• Dyer Matthew
• Reed Brian

Classifications

• G01N33/6842

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title

• G01N33/6818 Primary

  Sequencing of polypeptides · CPC title

• G01N33/54366

  Apparatus specially adapted for solid-phase testing · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2525/205

  Aptamer · CPC title